Acute Infection Model Research Articles

Design, synthesis and biological evaluation of N-oxide derivatives with potent in vivo antileishmanial activity.

Leishmaniasis is a neglected disease that affects 12 million people living mainly in developing countries. Herein, 24 new N-oxide-containing compounds were synthesized followed by in vitro and in vivo evaluation of their antileishmanial activity. Compound 4f, a furoxan derivative, was particularly remarkable in this regard, with EC50 value of 3.6 μM against L. infantum amastigote forms and CC50 value superior to 500 μM against murine peritoneal macrophages. In vitro studies suggested that 4f may act by a dual effect, by releasing nitric oxide after biotransformation and by inhibiting cysteine protease CPB (IC50: 4.5 μM). In vivo studies using an acute model of infection showed that compound 4f at 7.7 mg/Kg reduced ~90% of parasite burden in the liver and spleen of L. infantum-infected BALB/c mice. Altogether, these outcomes highlight furoxan 4f as a promising compound for further evaluation as an antileishmanial agent.

Analogs of Marinopyrrole A Show Enhancement to Observed In Vitro Potency against Acute Toxoplasma gondii Infection.

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a globally distributed infection with severe clinical consequences for immunocompromised individuals and developing fetuses. There are few available treatments, and these are associated with potentially severe adverse effects. Marinopyrrole A, a compound discovered in a marine Streptomyces species, has previously been found to exhibit potent antimicrobial activity, prompting our interest in exploring efficacy against Toxoplasma gondii. We found that marinopyrrole A was a highly potent anti-Toxoplasma molecule, with an in vitro 50% maximal inhibitory concentration (IC50) of 0.31 μM, corresponding to a higher potency than that of the current standard of care (pyrimethamine); however, addition of 20% serum led to abrogation of potency, and toxicity to human cell lines was observed. Yet, application of marinopyrrole A to an in vivo lethal acute infection model facilitated significantly enhanced survival at doses of 5, 10, and 20 mg/kg. We then tested a series of marinopyrrole A analogs (RL002, RL003, and RL125) and demonstrated significantly increased potency in vitro, with IC50 values ranging from 0.09 to 0.17 μM (3.6- to 6.8-fold increase relative to pyrimethamine). No detectable cytotoxicity was observed up to 50 μM in human foreskin fibroblasts, with cytotoxicity in HepG2 cells ranging from ∼28 to 50 μM, corresponding to >200-fold selectivity for parasites over host cells. All analogs additionally showed reduced sensitivity to serum. Further, RL003 potently inhibited in vitro-generated bradyzoites at 0.245 μM. Taken together, these data support further development of marinopyrrole A analogs as promising anti-Toxoplasma molecules to further combat this prevalent infection.

Increased systemic RNA oxidative damage and diagnostic value of RNA oxidative metabolites during Shigella flexneri-induced intestinal infection.

BACKGROUND Shigella flexneri (S. flexneri) is a major pathogen causing acute intestinal infection, but the systematic oxidative damage incurred during the course of infection has not been investigated.AIMTo investigate the incurred systemic RNA oxidative damage and the diagnostic value of RNA oxidative metabolites during S. flexneri-induced intestinal infection.METHODSIn this study, a Sprague-Dawley rat model of acute intestinal infection was established by oral gavage with S. flexneri strains. The changes in white blood cells (WBCs) and cytokine levels in blood and the inflammatory response in the colon were investigated. We also detected the RNA and DNA oxidation in urine and tissues.RESULTS S. flexneri infection induced an increase in WBCs, C-reactive protein, interleukin (IL)-6, IL-10, IL-1β, IL-4, IL-17a, IL-10, and tumor necrosis factor α (TNF-α) in blood. Of note, a significant increase in urinary 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn), an important marker of total RNA oxidation, was detected after intestinal infection (P = 0.03). The urinary 8-oxo-Gsn level returned to the baseline level after recovery from infection. In addition, the results of a correlation analysis showed that urinary 8-oxo-Gsn was positively correlated with the WBC count and the cytokines IL-6, TNF-α, IL-10, IL-1β, and IL-17α. Further detection of the oxidation in different tissues showed that S. flexneri infection induced RNA oxidative damage in the colon, ileum, liver, spleen, and brain.CONCLUSIONAcute infection induced by S. flexneri causes increased RNA oxidative damage in various tissues (liver, spleen, and brain) and an increase of 8-oxo-Gsn, a urinary metabolite. Urinary 8-oxo-Gsn may be useful as a biomarker for evaluating the severity and prognosis of infection.

RSV Promotes Epithelial Neuroendocrine Phenotype Differentiation through NODAL Signaling Pathway.

Background Respiratory syncytial virus (RSV) infects infants and children, predisposing them to development of asthma during adulthood. Epithelial neuroendocrine phenotypes may be associated with development of asthma. This study hopes to ascertain if RSV infection promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway. Methods The GSE6802 data set was obtained from the GEO database, and the differential genes were analyzed using the R language. An in vitro model was constructed with RSV infected human respiratory epithelial cells, and then real-time qPCR and immunofluorescence were used to detect the expression of different epithelial biomarkers and airway neuropeptides. The acute and chronic infection model of RSV infection was established by intranasal injection of RSV into guinea pigs. Immunohistochemistry and Western blot were used to detect the expression of pulmonary neuroendocrine cells markers ENO2 and neuropeptides. Results The expression levels of ENO2, SP, CGRP, and NODAL/ACTRII were significantly higher in the RSV infection group than those of the control group, which were abrogated by siRNA-NODAL. In vivo, we found that the expression levels of ENO2, SP, and CGRP were significantly higher than that of the control group. Conclusion RSV promotes epithelial neuroendocrine phenotypes through the NODAL signaling pathway.

PD-1 Blockade Restores the Proliferation of Peripheral Blood Lymphocyte and Inhibits Lymphocyte Apoptosis in a BALB/c Mouse Model of CP BVDV Acute Infection.

Acute infection of bovine viral diarrhea virus (BVDV) is associated with immune dysfunction and can cause peripheral blood lymphopenia and lymphocyte apoptosis. Our previous study has confirmed that programmed death-1 (PD-1) blockade inhibits peripheral blood lymphocytes (PBL) apoptosis and restores proliferation and anti-viral immune functions of lymphocytes after BVDV infection in vitro. However, the situation in vivo remains to be further studied and confirmed. Therefore, in this study, we established a BALB/c mouse model of acute BVDV infection with cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain NY-1). Then, we examined the mRNA and protein levels of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC) from BVDV-infected mice and analyzed the effects of PD-1 blockade on the proportions of CD3+, CD4+, and CD8+ T cell subsets, the apoptosis and proliferation of PBL, and the production of IL-2 and IFN-γ. We found that leukopenia, lymphocytopenia, and thrombocytopenia were developed in both CP and NCP BVDV-infected mice at day 7 of post-infection. The mRNA and protein expression of PD-1 and PD-L1 were significantly upregulated in CP and NCP BVDV-infected mice. Moreover, PD-1/PD-L1 upregulation was accompanied by leukopenia and lymphopenia. Additionally, PD-1 blockade inhibited PBL apoptosis and virus replication, restored the proportions of CD3+, CD4+, and CD8+ T cell subsets, and increased IFN-γ production and p-ERK expression in BVDV-infected mice. However, blocking PD-1 did not significantly affect PBL proliferation and IL-2 production in NCP BVDV-infected mice. Our findings further confirmed the immunomodulatory role of PD-1 in peripheral blood lymphocytopenia in vivo and provided a scientific basis for exploring the molecular mechanism of immune dysfunction caused by acute BVDV infection.

From the Editor’s Desk…

From the Editor’s Desk…

Chemical Fingerprint Profiles and Pharmacodynamic Investigation for Quality Evaluation of Moxa Smoke by UHPLC in a Rat Model of Superficial Infection

Introduction Moxibustion, a traditional Chinese medicine technique, involves the use of moxa smoke from Folium Artemisia argyi to treat various disorders, especially superficial infections. However, there is a higher health risk for people exposed to high levels of moxa smoke for extended durations. Here, we report the first ultra-high-performance liquid chromatography (UHPLC) fingerprint profiles and pharmacodynamic evaluation of moxa smoke, as well as evaluation of its aqueous solution on a rat model of superficial infection. Methods A novel method for moxa smoke fingerprint profiling was developed using UHPLC under characteristic wavelength. Chromatographic peaks were further analyzed by ultra-high-performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF/MS). 12 sample batches obtained from various Chinese provinces were then analyzed using similarity evaluation, clustering analysis, and principal component analysis. The pharmacodynamics of moxa smoke and moxa aqueous solution were investigated on a rat model of acute skin wound infection. Results UHPLC fingerprint profiles of 12 batches of moxa smoke were generated at 270 nm wavelength and 21 chromatographic peaks extracted as common peaks. Similarity between the 12 batches ranged from 0.341 to 0.982. Based on cluster analysis, the 12 batches of moxa smoke samples were clustered into five groups. Principal component analysis showed that the cumulative contribution of the three principal components reached 90.17%. Eigenvalues of the first, second, and third principal components were 10.794, 6.504, and 1.638, respectively. The corresponding variance contribution rates were 51.40%, 30.97%, and 7.80%, respectively. Pharmacological analysis found that wound healing was slow in the model group relative to the mupirocin ointment, moxa smoke, and aqueous moxa smoke solution groups. Histological analysis revealed markedly reduced tissue inflammation in rats treated with moxa smoke or its aqueous solution. Conclusions Moxa smoke and its aqueous solution significantly promote wound healing upon superficial infection. A novel quality control method for moxa smoke was established and evaluated for the first time. As its main effects are unchanged, the transformation of moxa smoke into aqueous moxa smoke improves safety and is a simple and controllable process.

Nox2-derived superoxide radical is crucial to control acute Trypanosoma cruzi infection

Trypanosoma cruzi is a flagellated protozoan that undergoes a complex life cycle between hematophagous insects and mammals. In humans, this parasite causes Chagas disease, which in thirty percent of those infected, would result in serious chronic pathologies and even death. Macrophages participate in the first stages of infection, mounting a cytotoxic response which promotes massive oxidative damage to the parasite. On the other hand, T. cruzi is equipped with a robust antioxidant system to repeal the oxidative attack from macrophages. This work was conceived to explicitly assess the role of mammalian cell-derived superoxide radical in a murine model of acute infection by T. cruzi. Macrophages derived from Nox2-deficient (gp91phox-/-) mice produced marginal amounts of superoxide radical and were more susceptible to parasite infection than those derived from wild type (wt) animals. Also, the lack of superoxide radical led to an impairment of parasite differentiation inside gp91phox-/- macrophages. Biochemical or genetic reconstitution of intraphagosomal superoxide radical formation in gp91phox-/- macrophages reverted the lack of control of infection. Along the same line, gp91phox-/- infected mice died shortly after infection. In spite of the higher lethality, parasitemia did not differ between gp91phox-/- and wt animals, recapitulating an observation that has led to conflicting interpretations about the importance of the mammalian oxidative response against T. cruzi. Importantly, gp91phox-/- mice presented higher and disseminated tissue parasitism, as evaluated by both qPCR- and bioimaging-based methodologies. Thus, this work supports that Nox2-derived superoxide radical plays a crucial role to control T. cruzi infection in the early phase of a murine model of Chagas disease.

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells.

SummaryB cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity.

Resolution of hepatitis E virus infection in CD8+ T cell-depleted rhesus macaques

HEV is a significant cause of acute hepatitis globally. Some genotypes establish persistent infection when immunity is impaired. Adaptive immune mechanisms that mediate resolution of infection have not been identified. Herein, the requirement for CD8+ T cells to control HEV infection was assessed in rhesus macaques, a model of acute and persistent HEV infection in humans. Rhesus macaques were untreated or treated with depleting anti-CD8α monoclonal antibodies before challenge with an HEV genotype (gt)3 isolate derived from a chronically infected human patient. HEV replication, alanine aminotransferase, anti-capsid antibody and HEV-specific CD4+ and CD8+ T cell responses were assessed after infection. HEV control in untreated macaques coincided with the onset of a neutralizing IgG response against the ORF2 capsid and liver infiltration of functional HEV-specific CD4+ and CD8+ T cells. Virus control was delayed by 1 week in CD8+ T cell-depleted macaques. Infection resolved with onset of a neutralizing IgG antibody response and a much more robust expansion of CD4+ T cells with antiviral effector function. Liver infiltration of functional CD8+ T cells coincident with HEV clearance in untreated rhesus macaques, and a 1-week delay in HEV clearance in CD8+ T cell-depleted rhesus macaques, support a role for this subset in timely control of virus replication. Resolution of infection in the absence of CD8+ T cells nonetheless indicates that neutralizing antibodies and/or CD4+ T cells may act autonomously to inhibit HEV replication. HEV susceptibility to multiple adaptive effector mechanisms may explain why persistence occurs only with generalized immune suppression. The findings also suggest that neutralizing antibodies and/or CD4+ T cells should be considered as a component of immunotherapy for chronic infection. The hepatitis E virus (HEV) is a major cause of liver disease globally. Some genetic types (genotypes) of HEV persist in the body if immunity is impaired. Our objective was to identify immune responses that promote clearance of HEV. Findings indicate that HEV may be susceptible to multiple arms of the immune response that can act independently to terminate infection. They also provide a pathway to assess immune therapies for chronic HEV infection.

Engagement of ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells

Abstract Resident memory T cells (TRM) provide localized protective immunity in non-lymphoid tissues, but the signals and interactions within non-lymphoid tissues involved in generating TRM are incompletely defined. Inducible T-cell costimulator (ICOS) is a costimulatory molecule with well elaborated roles in regulating differentiation of CD4+ T cell populations, its function in CD8+ T cells is largely unexplored. With acute infection models, we found that ICOS-deficient CD8+ T cells showed minimal defects in the initial effector expansion and the generation of recirculating memory subsets. However, consistent with its high expression in TRM, ICOS deficiency significantly compromised the establishment of CD8+ TRM cells in diverse non-lymphoid tissues. We further confirmed that rather than pre-programming the TRM fate during initial T cell priming, the interaction between ICOS ligand (ICOSL) and ICOS in the non-lymphoid tissues during CD8+ T cell recruitment is critical for efficient tissue retention and acquisition of tissue residency signatures. ICOS signaling through PI3K pathway is key for TRM generation and ICOS stimulation promotes downregulation of KLF2, a transcription factor that regulates T cell trafficking, in CD8+ T cells after entering non-lymphoid tissues. Altogether, our data illustrate that engagement of ICOS is one of the critical local costimulatory cues that promote the establishment of tissue-resident populations, with potential implication for therapeutic manipulation.

Establishment of infection mode and Penaeus monodon hemocytes transcriptomics analysis under decapod iridescent virus 1 (DIV1) challenge

With the increasing density of shrimp breeding, environmental deterioration and stress associated with intensification have caused diseases occurred frequently. Due to the high fatality rate of decapod iridescent virus 1 (DIV1), a new type of virus discovered in shrimp in recent years, it has caused serious damage to the shrimp farming industry. Penaeus monodon is one of the commonly cultivated species of prawns in the world. In the current study, LD50 was calculated to evaluate the virulence of DIV1 and the DIV1 copies in five tissues were quantitatively detected, which proved that P. monodon could be a host of DIV1. On these basis, a stable and effective DIV1 infection (including latent infection and acute infection) model was established for the first time. In addition, RNA-Seq platform and bioinformatics analysis were used to investigate the hemocyte immune response to DIV1 infection in P. monodon. A total of 31,657 unigenes were find significant assembled when compared to known sequences in major databases, and a total of 29,820 SSRs were obtained in the hemocytes transcriptome. A total of 6236 differentially expressed genes (DEGs) were identified, including 3140 up-regulated genes and 3096 down-regulated genes. Through the KEGG pathway enrichment analysis of DEGs, six immune-related pathways were enriched significantly, including Toll and Imd signaling pathway, PI3K-Akt signaling pathway, NF-kappa B signaling pathway, NOD-like receptor signaling pathway, Lysosome and MAPK signaling pathway. Moreover, many DEGs associated with Cholesterol metabolism, Pyruvate metabolism, Arginine and proline metabolism, Nicotinate and nicotinamide metabolism, Pyrimidine metabolism, Tyrosine metabolism and Alanine, aspartate and glutamate metabolism were also significantly modified. Moreover, Glycolysis / Gluconeogenesis and Pentose phosphate pathway, as the markers of Warburg effect, were significantly up-regulated after DIV1 infection. This study provides an ideal infection model for studying the molecular mechanism of P. monodon from the latent infection stage to the acute infection stage, initially reveals the molecular mechanism of DIV1 infection in P. monodon and provides valuable genetic resources and research methods for following molecular marker development and screening the families of P. monodon resistant to DIV1. The ultimate goal is to develop methods to avoid disease outbreaks in farmed shrimp.

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

Identification of an orally active carbazole aminoalcohol derivative with broad-spectrum anti-animal trypanosomiasis activity

Animal trypanosomiasis, caused by the members of subgenus Trypanozoon (Trypanosoma brucei brucei, T. evansi and T. equiperdum), has reduced animal productivity leading to significant negative economic impacts in endemic regions. Due to limited drug discovery and the emergence of drug-resistance over many recent decades, novel and effective compounds against animal trypanosomiasis are urgently required. This study was conducted to evaluate the antitrypanosomal potential of a batch of carbazole aminoalcohol derivatives. Among them, we found that the most effective compound was H1402, which exhibited potent trypanocidal efficacy against the bloodstream-form of T. b. brucei (EC50 = 0.73 ± 0.05 µM) and presented low cytotoxicity against two mammalian cell lines with CC50 > 30 µM. Using a murine model of acute infection, oral administration with H1402 demonstrated a complete clearance of T. b. brucei and all the infected mice were cured when they were treated twice daily for 5 days at a dose of 100 mg/kg. Furthermore, parasites were not detected in mice infected with T. evansi and T. equiperdum (the causative agents of surra and dourine, respectively, in animals) within 30 days following the same regimen with H1402. In addition, H1402 caused severe morphological and ultrastructural destruction to trypanosomes, as well as causing phosphatidylserine externalization, which are suggested to be the most likely cause of cell death. Overall, the present data demonstrated that H1402 could be promising as a rapid, safe and orally active lead compound for the development of new chemotherapeutics for animal trypanosomiasis.

Environmental and clinical isolates of Herbaspirillum induce pulmonary infection in mice and its secretome is cytotoxic to human lung cells.

Introduction. In recent years, the Herbaspirillum genus has emerged as a pathogen in healthcare-related infections and has became stablished as an opportunistic pathogen.Hypothesis/Gap Statement. Little is known about the pathogenesis induced by Herbaspirillum genus.Aim. To evaluate the cytotoxic effects of genus Herbaspirillum, its ability to adhere to lung human cells and the ability of environmental and clinical strains of Herbaspirillum to induce pneumonia in mice.Methodology. Environmental and clinical isolates of Herbaspirillum were examined for their cytotoxic effects on the Calu-3 cell lineage. Cytotoxic activity of secretome was tested using MTT/neutral red assays and cell morphology analysis. Herbaspirillum adhesion on Calu-3 cells was assessed using bright-field microscopy and cell-associated bacteria were counted. A mouse model of acute lung infection was done using a clinical and an environmental strain. Adult male mice were used, and the pneumonia was inducted by intra-tracheal inoculation of 108 or 109 bacteria. Mice weight variations were evaluated at the end of the experiment. Bronchoalveolar lavage was collected and evaluated for total and differential cytology. A histological examination of lungs was performed giving a histological score.Results. The secretomes of all the strains induced morphological alterations in cells, but only H. seropedicae SmR1 were cytotoxic in MTT and neutral red assays. Clinical strains of H. frisingense AU14459 and H. hutttiense subsp. huttiense AU11883 exhibited low adherence to lung cells, while SmR1 was non-adhesive. Following intratracheal inoculation, mice treated with 109 c.f.u. of the SmR1 and AU11883 strains lost 18 and 6% of their weight over 7 days, respectively, and presented moderate clinical signs. Infected mice showed inflammatory cell infiltration in the perivascular and peribroncheal/peribronchiolar spaces. Bronchoalveolar fluid of mice inoculated with SmR1 109 c.f.u. presented an increase in total leucocyte cells and in neutrophils population.Conclusion. These in vivo and in vitro results provide insights into how some Herbaspirillum strains cause infection in humans, providing a basis for the characterization of pathogenesis studies on this emerging infectious agent.

Itraconazole for COVID-19: preclinical studies and a proof-of-concept randomized clinical trial

BackgroundThe antifungal drug itraconazole exerts in vitro activity against SARS-CoV-2 in Vero and human Caco-2 cells. Preclinical and clinical studies are required to investigate if itraconazole is effective for the treatment and/or prevention of COVID-19. MethodsDue to the initial absence of preclinical models, the effect of itraconazole was explored in a clinical, proof-of-concept, open-label, single-center study, in which hospitalized COVID-19 patients were randomly assigned to standard of care with or without itraconazole. Primary outcome was the cumulative score of the clinical status until day 15 based on the 7-point ordinal scale of the World Health Organization. In parallel, itraconazole was evaluated in a newly established hamster model of acute SARS-CoV-2 infection and transmission, as soon as the model was validated. FindingsIn the hamster acute infection model, itraconazole did not reduce viral load in lungs, stools or ileum, despite adequate plasma and lung drug concentrations. In the transmission model, itraconazole failed to prevent viral transmission. The clinical trial was prematurely discontinued after evaluation of the preclinical studies and because an interim analysis showed no signal for a more favorable outcome with itraconazole: mean cumulative score of the clinical status 49 vs 47, ratio of geometric means 1.01 (95% CI 0.85 to 1.19) for itraconazole vs standard of care. InterpretationDespite in vitro activity, itraconazole was not effective in a preclinical COVID-19 hamster model. This prompted the premature termination of the proof-of-concept clinical study.Funding: KU Leuven, Research Foundation - Flanders (FWO), Horizon 2020, Bill and Melinda Gates Foundation

Modeling the complete kinetics of coxsackievirus B3 reveals human determinants of host-cell feedback.

Complete kinetic models are pervasive in chemistry but lacking in biological systems. We encoded the complete kinetics of infection for coxsackievirus B3 (CVB3), a compact and fast-acting RNA virus. The model consists of separable, detailed modules describing viral binding-delivery, translation-replication, and encapsidation. Specific module activities are dampened by the type I interferon response to viral double-stranded RNAs (dsRNAs), which is itself disrupted by viral proteinases. The experimentally validated kinetics uncovered that cleavability of the dsRNA transducer mitochondrial antiviral signaling protein (MAVS) becomes a stronger determinant of viral outcomes when cells receive supplemental interferon after infection. Cleavability is naturally altered in humans by a common MAVS polymorphism, which removes a proteinase-targeted site but paradoxically elevates CVB3 infectivity. These observations are reconciled with a simple nonlinear model of MAVS regulation. Modeling complete kinetics is an attainable goal for small, rapidly infecting viruses and perhaps viral pathogens more broadly. A record of this paper's transparent peer review process is included in the Supplemental information.

A SNP in the Cache 1 Signaling Domain of Diguanylate Cyclase STM1987 Leads to Increased In Vivo Fitness of Invasive Salmonella Strains.

Nontyphoidal Salmonella (NTS) strains are associated with gastroenteritis worldwide but are also the leading cause of bacterial bloodstream infections in sub-Saharan Africa. The invasive NTS (iNTS) strains that cause bloodstream infections differ from standard gastroenteritis-causing strains by >700 single-nucleotide polymorphisms (SNPs). These SNPs are known to alter metabolic pathways and biofilm formation and to contribute to serum resistance and are thought to signify iNTS strains becoming human adapted, similar to typhoid fever-causing Salmonella strains. Identifying SNPs that contribute to invasion or increased virulence has been more elusive. In this study, we identified a SNP in the cache 1 signaling domain of diguanylate cyclase STM1987 in the invasive Salmonella enterica serovar Typhimurium type strain D23580. This SNP was conserved in 118 other iNTS strains analyzed and was comparatively absent in global S Typhimurium isolates associated with gastroenteritis. STM1987 catalyzes the formation of bis-(3',5')-cyclic dimeric GMP (c-di-GMP) and is proposed to stimulate production of cellulose independent of the master biofilm regulator CsgD. We show that the amino acid change in STM1987 leads to a 10-fold drop in cellulose production and increased fitness in a mouse model of acute infection. Reduced cellulose production due to the SNP led to enhanced survival in both murine and human macrophage cell lines. In contrast, loss of CsgD-dependent cellulose production did not lead to any measurable change in in vivo fitness. We hypothesize that the SNP in stm1987 represents a pathoadaptive mutation for iNTS strains.

Combination With Tomatidine Improves the Potency of Posaconazole Against Trypanosoma cruzi.

Azoles such as posaconazole (Posa) are highly potent against Trypanosoma cruzi. However, when tested in chronic Chagas disease patients, a high rate of relapse after Posa treatment was observed. It appears that inhibition of T. cruzi cytochrome CYP51, the target of azoles, does not deliver sterile cure in monotherapy. Looking for suitable combination partners of azoles, we have selected a set of inhibitors of sterol and sphingolipid biosynthetic enzymes. A small-scale phenotypic screening was conducted in vitro against the proliferative forms of T. cruzi, extracellular epimastigotes and intracellular amastigotes. Against the intracellular, clinically relevant forms, four out of 15 tested compounds presented higher or equal activity as benznidazole (Bz), with EC50 values ≤2.2 μM. Ro48-8071, an inhibitor of lanosterol synthase (ERG7), and the steroidal alkaloid tomatidine (TH), an inhibitor of C-24 sterol methyltransferase (ERG6), exhibited the highest potency and selectivity indices (SI = 12 and 115, respectively). Both were directed to combinatory assays using fixed-ratio protocols with Posa, Bz, and fexinidazole. The combination of TH with Posa displayed a synergistic profile against amastigotes, with a mean ΣFICI value of 0.2. In vivo assays using an acute mouse model of T. cruzi infection demonstrated lack of antiparasitic activity of TH alone in doses ranging from 0.5 to 5 mg/kg. As observed in vitro, the best combo proportion in vivo was the ratio 3 TH:1 Posa. The combination of Posa at 1.25 mpk plus TH at 3.75 mpk displayed suppression of peak parasitemia of 80% and a survival rate of 60% in the acute infection model, as compared to 20% survival for Posa at 1.25 mpk alone and 40% for Posa at 10 mpk alone. These initial results indicate a potential for the combination of posaconazole with tomatidine against T. cruzi.

Fluoxetine Can Inhibit SARS-CoV-2 In Vitro.

An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resulted in the coronavirus disease pandemic, drastically affecting global health and economy. Though the understanding of the disease has improved, fighting the virus remains challenging. One of the strategies is repurposing existing drugs as inhibitors of SARS-CoV-2. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, reportedly inhibits the replication of RNA viruses, especially Coxsackieviruses B (CVB), such as CV-B4 in vitro and in vivo. Therefore, in this study, we investigated the in vitro antiviral activity of FLX against SARS-CoV-2 in a model of acute infection. When 10 μM of FLX was added to SARS-CoV-2-infected Vero E6 cells, the virus-induced cytopathic effect was not observed. In this model, the level of infectious particles in the supernatant was lower than that in controls. The level was below the limit of detection of the assay up to day 3 post-infection when FLX was administered before viral inoculation or simultaneously followed by daily inoculation. In conclusion, FLX can inhibit SARS-CoV-2 in vitro. Further studies are needed to investigate the potential value of FLX to combat SARS-CoV-2 infections, treat SARS-CoV-2-induced diseases, and explain the antiviral mechanism of this molecule to pave way for novel treatment strategies.