Further preclinical characterization of molnupiravir against SARS-CoV-2: Antiviral activity determinants and viral genome alteration patterns.

The SARS-CoV-2 pandemic has highlighted the need for broad-spectrum antiviral drugs to respond promptly to viral emergence. We conducted a preclinical study of molnupiravir (MOV) against SARS-CoV-2 to fully characterise its antiviral properties and mode of action. The antiviral activity of different concentrations of MOV was evaluated ex vivo on human airway epithelium (HAE) and in vivo in a hamster model at three escalating doses (150, 300 and 400 mg/kg/day) according to three different regimens (preventive, pre-emptive and curative). We assessed viral loads and infectious titres at the apical pole of HAE and in hamster lungs, and MOV trough concentration in plasma and lungs. To explore the mode of action of the MOV, the entire genomes of the collected viruses were deep-sequenced. MOV effectively reduced viral titres in HAE and in the lungs of treated animals. Early treatment after infection was a key factor in efficacy, probably associated with high lung concentrations of MOV, suggesting good accumulation in the lung. MOV induced genomic alteration in viral genomes with an increase in the number of minority variants, and predominant G to A transitions. The observed reduction in viral replication and its mechanism of action leading to lethal mutagenesis, supported by clinical trials showing antiviral action in humans, provide a convincing basis for further research as an additional means in the fight against COVID-19 and other RNA viruses.

Pre-clinical evaluation of antiviral activity of nitazoxanide against SARS-CoV-2.

BACKGROUND: To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy. METHODS: In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2. FINDINGS: First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50. INTERPRETATION: These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19. FUNDING: This work was supported by the Fondation de France "call FLASH COVID-19", project TAMAC, by "Institut national de la santé et de la recherche médicale" through the REACTing (REsearch and ACTion targeting emerging infectious diseases), by REACTING/ANRS MIE under the agreement No. 21180 ('Activité des molécules antivirales dans le modèle hamster'), by European Virus Archive Global (EVA 213 GLOBAL) funded by the European Union's Horizon 2020 research and innovation program under grant agreement No. 871029 and DNDi under support by the Wellcome Trust Grant ref: 222489/Z/21/Z through the COVID-19 Therapeutics Accelerator".

The SARS-CoV-2 Alpha variant exhibits comparable fitness to the D614G strain in a Syrian hamster model.

Late 2020, SARS-CoV-2 Alpha variant emerged in United Kingdom and gradually replaced G614 strains initially involved in the global spread of the pandemic. In this study, we use a Syrian hamster model to compare a clinical strain of Alpha variant with an ancestral G614 strain. The Alpha variant succeed to infect animals and to induce a pathology that mimics COVID-19. However, both strains replicate to almost the same level and induced a comparable disease and immune response. A slight fitness advantage is noted for the G614 strain during competition and transmission experiments. These data do not corroborate the epidemiological situation observed during the first half of 2021 in humans nor reports that showed a more rapid replication of Alpha variant in human reconstituted bronchial epithelium. This study highlights the need to combine data from different laboratories using various animal models to decipher the biological properties of newly emerging SARS-CoV-2 variants.

Hydroxychloroquine and azithromycin used alone or combined are not effective against SARS-CoV-2 ex vivo and in a hamster model.

Drug repositioning has been used extensively since the beginning of the COVID-19 pandemic in an attempt to identify antiviral molecules for use in human therapeutics. Hydroxychloroquine and azithromycin have shown inhibitory activity against SARS-CoV-2 replication in different cell lines. Based on such in vitro data and despite the weakness of preclinical assessment, many clinical trials were set up using these molecules. In the present study, we show that hydroxychloroquine and azithromycin alone or combined does not block SARS-CoV-2 replication in human bronchial airway epithelia. When tested in a Syrian hamster model, hydroxychloroquine and azithromycin administrated alone or combined displayed no significant effect on viral replication, clinical course of the disease and lung impairments, even at high doses. Hydroxychloroquine quantification in lung tissues confirmed strong exposure to the drug, above in vitro inhibitory concentrations. Overall, this study does not support the use of hydroxychloroquine and azithromycin as antiviral drugs for the treatment of SARS-CoV-2 infections.

Sensory neuron-derived TAFA4 promotes macrophage tissue repair functions.

Inflammation is a defence response to tissue damage that requires tight regulation in order to prevent impaired healing. Tissue-resident macrophages have a key role in tissue repair1, but the precise molecular mechanisms that regulate the balance between inflammatory and pro-repair macrophage responses during healing remain poorly understood. Here we demonstrate a major role for sensory neurons in promoting the tissue-repair function of macrophages. In a sunburn-like model of skin damage in mice, the conditional ablation of sensory neurons expressing the Gαi-interacting protein (GINIP) results in defective tissue regeneration and in dermal fibrosis. Elucidation of the underlying molecular mechanisms revealed a crucial role for the neuropeptide TAFA4, which is produced in the skin by C-low threshold mechanoreceptors-a subset of GINIP+ neurons. TAFA4 modulates the inflammatory profile of macrophages directly in vitro. In vivo studies in Tafa4-deficient mice revealed that TAFA4 promotes the production of IL-10 by dermal macrophages after UV-induced skin damage. This TAFA4-IL-10 axis also ensures the survival and maintenance of IL-10+TIM4+ dermal macrophages, reducing skin inflammation and promoting tissue regeneration. These results reveal a neuroimmune regulatory pathway driven by the neuropeptide TAFA4 that promotes the anti-inflammatory functions of macrophages and prevents fibrosis after tissue damage, and could lead to new therapeutic perspectives for inflammatory diseases.

Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model.

Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we use a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment is initiated before or simultaneously to infection, favipiravir has a strong dose effect, leading to reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlates with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. Antiviral efficacy is achieved with plasma drug exposure comparable with those previously found during human clinical trials. Notably, the highest dose of favipiravir tested is associated with signs of toxicity in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

ß2-adrenergic signals downregulate the innate immune response and reduce host resistance to viral infection.

In humans, psychological stress has been associated with a higher risk of infectious illness. However, the mechanisms by which the stress pathway interferes with host response to pathogens remain unclear. We demonstrate here a role for the ß2-adrenergic receptor (ß2-AR), which binds the stress mediators adrenaline and noradrenaline, in modulating host response to mouse cytomegalovirus (MCMV) infection. Mice treated with a ß2-AR agonist were more susceptible to MCMV infection. By contrast, ß2-AR deficiency resulted in a better clearance of the virus, less tissue damage, and greater resistance to MCMV. Mechanistically, we found a correlation between higher levels of IFN-γ production by liver natural killer (NK) cells and stronger resistance to MCMV. However, the control of NK cell IFN-γ production was not cell intrinsic, revealing a cell-extrinsic downregulation of the antiviral NK cell response by adrenergic neuroendocrine signals. This pathway reduces host immune defense, suggesting that the blockade of the ß2-AR signaling could be used to increase resistance to infectious diseases.

Tracking Dynamics of Spontaneous Tumors in Mice Using Photon-Counting Computed Tomography.

Computed tomography is a powerful medical imaging modality for longitudinal studies in cancer to follow neoplasia progression and evaluate anticancer therapies. Here, we report the generation of a photon-counting micro-computed tomography (PC-CT) method based on hybrid pixel detectors with enhanced sensitivity and precision of tumor imaging. We then applied PC-CT for longitudinal imaging in a clinically relevant liver cancer model, the Alb-R26Met mice, and found a remarkable heterogeneity in the dynamics for tumors at the initiation phases. Instead, the growth curve of evolving tumors exhibited a comparable exponential growth, with a constant doubling time. Furthermore, longitudinal PC-CT imaging in mice treated with a combination of MEK and BCL-XL inhibitors revealed a drastic tumor regression accompanied by a striking remodeling of macrophages in the tumor microenvironment. Thus, PC-CT is a powerful system to detect cancer initiation and progression, and to monitor its evolution during treatment.

Lupus Autoimmunity and Metabolic Parameters Are Exacerbated Upon High Fat Diet-Induced Obesity Due to TLR7 Signaling.

Systemic lupus erythematosus (SLE) patients have increased prevalence of metabolic syndrome but the underlying mechanisms are unknown. Toll-like receptor 7 (TLR7) that detects single stranded-RNA plays a key role in antimicrobial host defense and also contributes to the initiation and progression of SLE both in mice and humans. Here, we report the implication of TLR7 signaling in high fat diet (HFD)-induced metabolic syndrome and exacerbation of lupus autoimmunity in TLR8-deficient (TLR8ko) mice, which develop spontaneous lupus-like disease due to increased TLR7 signaling by dendritic cells (DCs). The aggravated SLE pathogenesis in HFD-fed TLR8ko mice was characterized by increased overall immune activation, anti-DNA autoantibody production, and IgG/IgM glomerular deposition that were coupled with increased kidney histopathology. Moreover, upon HFD TLR8ko mice developed metabolic abnormalities, including liver inflammation. In contrast, upon HFD TLR7/8ko mice did not develop SLE and both TLR7ko and TLR7/8ko mice were fully protected from metabolic abnormalities, including body weight gain, insulin resistance, and liver inflammation. Interestingly, HFD led to an increase of TLR7 expression in WT mice, that was coupled with increased TNF production by DCs, and this phenotype was more profound in TLR8ko mice. Our study uncovers the implication of TLR7 signaling in the interconnection of SLE and metabolic abnormalities, indicating that TLR7 might be a novel approach as a tailored therapy in SLE and metabolic diseases.

Vnn1 pantetheinase limits the Warburg effect and sarcoma growth by rescuing mitochondrial activity.

Like other tumors, aggressive soft tissue sarcomas (STS) use glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) for growth. Given the importance of the cofactor coenzyme A (CoA) in energy metabolism, we investigated the impact of Vnn1 pantetheinase-an enzyme that degrades pantetheine into pantothenate (vitamin B5, the CoA biosynthetic precursor) and cysyteamine-on tumor growth. Using two models, we show that Vnn1+ STS remain differentiated and grow slowly, and that in patients a detectable level of VNN1 expression in STS is associated with an improved prognosis. Increasing pantetheinase activity in aggressive tumors limits their growth. Using combined approaches, we demonstrate that Vnn1 permits restoration of CoA pools, thereby maintaining OXPHOS. The simultaneous production of cysteamine limits glycolysis and release of lactate, resulting in a partial inhibition of STS growth in vitro and in vivo. We propose that the Warburg effect observed in aggressive STS is reversed by induction of Vnn1 pantetheinase and the rewiring of cellular energy metabolism by its products.

Endogenous glucocorticoids control host resistance to viral infection through the tissue-specific regulation of PD-1 expression on NK cells.

Controlling the balance between immunity and immunopathology is crucial for host resistance to pathogens. After infection, activation of the hypothalamic-pituitary-adrenal (HPA) axis leads to the production of glucocorticoids. However, the pleiotropic effects of these steroid hormones make it difficult to delineate their precise role(s) in vivo. Here we found that the regulation of natural killer (NK) cell function by the glucocorticoid receptor (GR) was required for host survival after infection with mouse cytomegalovirus (MCMV). Mechanistically, endogenous glucocorticoids produced shortly after infection induced selective and tissue-specific expression of the checkpoint receptor PD-1 on NK cells. This glucocorticoid-PD-1 pathway limited production of the cytokine IFN-γ by spleen NK cells, which prevented immunopathology. Notably, this regulation did not compromise viral clearance. Thus, the fine tuning of NK cell functions by the HPA axis preserved tissue integrity without impairing pathogen elimination, which reveals a novel aspect of neuroimmune regulation.

Guanabenz Prevents d-Galactosamine/Lipopolysaccharide-Induced Liver Damage and Mortality.

Multi-organ failure in response to uncontrolled microbial infection is characterized by low blood pressure accompanied by a systemic over-inflammation state, caused by massive pro-inflammatory cytokines release and liver damage. Recently, the integrated stress response (ISR), characterized by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, was involved with controlling apoptosis in stressed hepatocytes and associated with poor survival to endotoxin challenge. Lipopolysaccharide (LPS) alone is able to induce the ISR in hepatocytes and can trigger massive liver damage along with tumor necrosis factor-alpha (TNF-α) expression. Consequently, drugs interfering with eIF2α phosphorylation may represent potential candidates for the treatment of such pathologies. We, therefore, used Guanabenz (GBZ), a small compound with enhancing eIF2α phosphorylation activity to evaluate its effect on bacterial LPS sensing and endotoxemia. GBZ is confirmed here to have an anti-inflammatory activity by increasing in vitro interleukin-10 (IL-10) production by LPS-stimulated dendritic cells. We further show that in the d-galactosamine (d-galN)/LPS-dependent lethality model, intraperitoneal injection of GBZ promoted mice survival, prevented liver damage, increased IL-10 levels, and inhibited TNF-α production. GBZ and its derivatives could therefore represent an interesting pharmacological solution to control systemic inflammation and associated acute liver failure.

In Vivo MRI Assessment of Hepatic and Splenic Disease in a Murine Model of Schistosomiasis [corrected].

BACKGROUND: Schistosomiasis (or bilharzia), a major parasitic disease, affects more than 260 million people worldwide. In chronic cases of intestinal schistosomiasis caused by trematodes of the Schistosoma genus, hepatic fibrosis develops as a host immune response to the helminth eggs, followed by potentially lethal portal hypertension. In this study, we characterized hepatic and splenic features of a murine model of intestinal schistosomiasis using in vivo magnetic resonance imaging (MRI) and evaluated the transverse relaxation time T2 as a non-invasive imaging biomarker for monitoring hepatic fibrogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CBA/J mice were imaged at 11.75 T two, six and ten weeks after percutaneous infection with Schistosoma mansoni. In vivo imaging studies were completed with histology at the last two time points. Anatomical MRI allowed detection of typical manifestations of the intestinal disease such as significant hepato- and splenomegaly, and dilation of the portal vein as early as six weeks, with further aggravation at 10 weeks after infection. Liver multifocal lesions observed by MRI in infected animals at 10 weeks post infection corresponded to granulomatous inflammation and intergranulomatous fibrosis with METAVIR scores up to A2F2. While most healthy hepatic tissue showed T2 values below 14 ms, these lesions were characterized by a T2 greater than 16 ms. The area fraction of increased T2 correlated (rS = 0.83) with the area fraction of Sirius Red stained collagen in histological sections. A continuous liver T2* decrease was also measured while brown pigments in macrophages were detected at histology. These findings suggest accumulation of hematin in infected livers. CONCLUSIONS/SIGNIFICANCE: Our multiparametric MRI approach confirms that this murine model replicates hepatic and splenic manifestations of human intestinal schistosomiasis. Quantitative T2 mapping proved sensitive to assess liver fibrogenesis non-invasively and may therefore constitute an objective imaging biomarker for treatment monitoring in diseases involving hepatic fibrosis.

Feline cutaneous mycobacteriosis: a review of clinical, pathological and molecular characterization of one case of Mycobacterium microti skin infection and nine cases of feline leprosy syndrome from France and New Caledonia.

BACKGROUND: Ten cats with skin lesions characteristic of cutaneous mycobacteriosis were included in this retrospective clinical, pathological and molecular study. HYPOTHESIS/OBJECTIVES: The aim of this study was to identify the causative agent and to compare the clinicopathological features of these cases with those of previous studies. METHODS: Cats were from the south east of France (eight cases), central France (one case) and New Caledonia (South Pacific; one case). Criteria for inclusion were histological evidence of granulomatous dermatitis and/or panniculitis, with acid-fast bacilli within macrophages or extracellularly in regions of tissue necrosis. PCR targeting the 16S-23S internal transcribed spacer region and sequence analysis were performed using DNA extracted from formalin-fixed, paraffin-embedded tissues from all cases. RESULTS: All cats were presented with a history of alopecic to ulcerated nodules. Most cases had limited disease, with one to few nodules, while others (three cats) showed a more aggressive clinical course. Lesions from eight cats yielded a sequence consistent with Mycobacterium lepraemurium, while Mycobacterium microti was identified postmortem from the cutaneous lesion in the cat originating from central France and euthanized for its debilitating condition. No PCR product could be amplified from the remaining specimen. CONCLUSIONS AND CLINICAL IMPORTANCE: Based on this geographically restricted case series, feline leprosy in southern France is most likely to be caused by M. lepraemurium and presents as a generally self-limiting disease. Molecular testing is essential to assess zoonotic potential, because M. microti-induced cutaneous mycobacteriosis can resemble feline leprosy syndrome.

Ulcerated and nonulcerated nontuberculous cutaneous mycobacterial granulomas in cats and dogs.

BACKGROUND: Mycobacterial granulomas of the skin and subcutis can be caused by one of a number of pathogens. This review concentrates on noncultivable species that cause diseases characterized by focal granuloma(s), namely leproid granuloma (in dogs) and feline leprosy (in cats). Clinically indistinguishable lesions can be caused by tuberculous organisms (Mycobacterium bovis and Mycobacterium microti) and members of the Mycobacterium avium complex. Rapidly growing mycobacterial species that cause infection of the subcutaneous panniculus associated with draining tracts are not discussed. Disease caused by Mycobacterium ulcerans is an important emerging differential diagnosis for ulcerated cutaneous nodules in certain localized regions. CLINICAL LESIONS: Lesions comprise one or multiple nodules in the skin/subcutis. These are generally firm and well circumscribed, and typically become denuded of hair. They may or may not ulcerate, depending on the virulence of the causal organisms and the immune response of the host. DIAGNOSIS: The most inexpensive, noninvasive means of diagnosis is by submission of methanol-fixed, Romanowsky-stained smears to a Mycobacterium Reference Laboratory after detecting negatively stained or acid-fast bacilli on cytological smears. Scrapings of material from slides usually provide sufficient mycobacterial DNA to enable identification of the causal organism using sequence analysis of amplicons after PCR using specific mycobacterial primers. THERAPY: Therapy relies upon a combination of marginal resection of easily accessible lesions and treatment using two or three drugs effective against slowly growing mycobacteria, choosing amongst rifampicin, clarithromycin, clofazimine and pradofloxacin/moxifloxacin.

Pythiosis in Africa.

We report the first case of pythiosis from Africa in an 8-month-old dog with a chronic and ulcerative cutaneous lesion. The etiologic agent belonged to the genus Pythium. Phylogenetic analysis placed the isolate in a sister group to the other P. insidiosum strains. However, the isolate may belong to a new Pythium species.