Preclinical in vivo assessment of the activity of AZD7442 anti-SARS-CoV-2 monoclonal antibodies against Omicron sublineages.

Therapeutic monoclonal antibodies have been successful in protecting vulnerable populations against SARS-CoV-2. However, their effectiveness has been hampered by the emergence of new variants. To adapt the therapeutic landscape, health authorities have based their recommendations mostly on in vitro neutralization tests. However, these do not provide a reliable understanding of the changes in the dose-effect relationship and how they may translate into clinical efficacy. Taking the example of EvusheldTM (AZD7442), we aimed to investigate how in vivo data can provide critical quantitative results and project clinical effectiveness. We used the Golden Syrian hamster model to estimate 90 % effective concentrations (EC90) of AZD7442 in vivo against SARS-CoV-2 Omicron BA.1, BA.2 and BA.5 variants. While our in vivo results confirmed the partial loss of AZD7442 activity for BA.1 and BA.2, they showed a much greater loss of efficacy against BA.5 than that obtained in vitro. We analyzed in vivo EC90s in perspective with antibody levels measured in a cohort of immunocompromised patients who received 300 mg of AZD7442. We found that a substantial proportion of patients had serum levels of anti-SARS-CoV-2 spike protein IgG above the estimated in vivo EC90 for BA.1 and BA.2 (21 % and 92 % after 1 month, respectively), but not for BA.5. These findings suggest that AZD7442 is likely to retain clinical efficacy against BA.2 and BA.1, but not against BA.5. Overall, the present study illustrates the importance of complementing in vitro investigations by preclinical studies in animal models to help predict the efficacy of monoclonal antibodies in humans.

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.

Need for a Standardized Translational Drug Development Platform: Lessons Learned from the Repurposing of Drugs for COVID-19.

In the absence of drugs to treat or prevent COVID-19, drug repurposing can be a valuable strategy. Despite a substantial number of clinical trials, drug repurposing did not deliver on its promise. While success was observed with some repurposed drugs (e.g., remdesivir, dexamethasone, tocilizumab, baricitinib), others failed to show clinical efficacy. One reason is the lack of clear translational processes based on adequate preclinical profiling before clinical evaluation. Combined with limitations of existing in vitro and in vivo models, there is a need for a systematic approach to urgent antiviral drug development in the context of a global pandemic. We implemented a methodology to test repurposed and experimental drugs to generate robust preclinical evidence for further clinical development. This translational drug development platform comprises in vitro, ex vivo, and in vivo models of SARS-CoV-2, along with pharmacokinetic modeling and simulation approaches to evaluate exposure levels in plasma and target organs. Here, we provide examples of identified repurposed antiviral drugs tested within our multidisciplinary collaboration to highlight lessons learned in urgent antiviral drug development during the COVID-19 pandemic. Our data confirm the importance of assessing in vitro and in vivo potency in multiple assays to boost the translatability of pre-clinical data. The value of pharmacokinetic modeling and simulations for compound prioritization is also discussed. We advocate the need for a standardized translational drug development platform for mild-to-moderate COVID-19 to generate preclinical evidence in support of clinical trials. We propose clear prerequisites for progression of drug candidates for repurposing into clinical trials. Further research is needed to gain a deeper understanding of the scope and limitations of the presented translational drug development platform.

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".

A new strategy to count and sort neutrophil-derived extracellular vesicles: Validation in infectious disorders.

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil-derived extracellular vesicles (NDEVs) involved in immune and thrombo-inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID-19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

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.

Investigation of Bufavirus and Parvovirus 4 in Patients with Gastro-Enteritis from the South-East of France.

Bufavirus (BuV) and human parvovirus 4 (PARV4) belong to the Parvoviridae family. We assessed BuV and PARV4 DNA presence by real-time PCR analysis in stool, blood and respiratory samples collected in patients from Marseille and Nice, two large cities in the South-East of France. Bu-V DNA was detected in diarrheic stool samples from 92 patients (3.6% of 2583 patients), particularly men and adults, and patients from the nephrology and the infectious disease departments. Among the patients with a BuV-positive stool sample and for whom at least one blood sample was available (n = 30 patients), BuV DNA was detected also in 3 blood samples. In contrast, BuV DNA was not detected in any of the respiratory samples from 23 patients with BuV-positive stool. BuV detection rate was comparable in stool samples from patients with and without diarrhea. We did not detect PARV4 DNA in any of the stool specimens (n = 2583 patients). Our results suggest that PARV4 fecal-oral transmission is rare or non-existent in the South-East of France while BuV circulates with a relatively high rate in this area.

Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2.

Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of imatinib in multiple models. Our results indicated that imatinib and another TKI, the masitinib, exhibit an antiviral activity in VeroE6 cells. However, imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of imatinib and similar TKIs as antivirals in the treatment of Covid-19.

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.

Novel CCM2 missense variants abrogating the CCM1-CCM2 interaction cause cerebral cavernous malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations mostly located within the central nervous system. Most deleterious variants are loss of function mutations in one of the three CCM genes. These genes code for proteins that form a ternary cytosolic complex with CCM2 as a hub. Very few CCM2 missense variants have been shown to be deleterious by modifying the ternary CCM complex stability. OBJECTIVES: To investigate the causality of novel missense CCM2 variants detected in patients with CCM. METHODS: The three CCM genes were screened in 984 patients referred for CCM molecular screening. Interaction between CCM1 and CCM2 proteins was tested using co-immunoprecipitation experiments for the CCM2 missense variants located in the phosphotyrosine binding (PTB) domain. RESULTS: 11 distinct CCM2 rare missense variants were found. Six variants predicted to be damaging were located in the PTB domain, four of them were novel. When co-transfected with CCM1 in HEK293T cells, a loss of interaction between CCM1 and CCM2 was observed for all six variants. CONCLUSION: We showed, using co-immunoprecipitation experiments, that CCM2 missense variants located in the PTB domain were actually damaging by preventing the normal interaction between CCM1 and CCM2. These data are important for diagnosis and genetic counselling, which are challenging in patients harbouring such variants.

Estimating attributable mortality due to nosocomial infections acquired in intensive care units.

BACKGROUND: The strength of the association between intensive care unit (ICU)-acquired nosocomial infections (NIs) and mortality might differ according to the methodological approach taken. OBJECTIVE: To assess the association between ICU-acquired NIs and mortality using the concept of population-attributable fraction (PAF) for patient deaths caused by ICU-acquired NIs in a large cohort of critically ill patients. SETTING: Eleven ICUs of a French university hospital. DESIGN: We analyzed surveillance data on ICU-acquired NIs collected prospectively during the period from 1995 through 2003. The primary outcome was mortality from ICU-acquired NI stratified by site of infection. A matched-pair, case-control study was performed. Each patient who died before ICU discharge was defined as a case patient, and each patient who survived to ICU discharge was defined as a control patient. The PAF was calculated after adjustment for confounders by use of conditional logistic regression analysis. RESULTS: Among 8,068 ICU patients, a total of 1,725 deceased patients were successfully matched with 1,725 control patients. The adjusted PAF due to ICU-acquired NI for patients who died before ICU discharge was 14.6% (95% confidence interval [CI], 14.4%-14.8%). Stratified by the type of infection, the PAF was 6.1% (95% CI, 5.7%-6.5%) for pulmonary infection, 3.2% (95% CI, 2.8%-3.5%) for central venous catheter infection, 1.7% (95% CI, 0.9%-2.5%) for bloodstream infection, and 0.0% (95% CI, -0.4% to 0.4%) for urinary tract infection. CONCLUSIONS: ICU-acquired NI had an important effect on mortality. However, the statistical association between ICU-acquired NI and mortality tended to be less pronounced in findings based on the PAF than in study findings based on estimates of relative risk. Therefore, the choice of methods does matter when the burden of NI needs to be assessed.

Early- and late-onset ventilator-associated pneumonia acquired in the intensive care unit: comparison of risk factors.

PURPOSE: To compare risk factors of early- (E) and late-onset (L) ventilator-associated pneumonia (VAP). MATERIALS AND METHODS: An epidemiological survey based on a nosocomial infection surveillance program of 11 intensive care units (ICUs) of university teaching hospitals in Lyon, France, was conducted. A total of 7236 consecutive ventilated patients, older than 18 years and hospitalized in ICUs for at least 48 hours, were studied between 1996 and 2002. Data during ICU stay, patient-dependent risk factors, device exposure, nosocomial infections occurrence, and outcome were collected. The cutoff point definition between E-VAP (six days) was based on the daily hazard rate of VAP. RESULTS: The VAP incidence rate was 13.1%, 356 (37.6%) E-VAP (within 6 days of admission) and 590 (62.4%) L-VAP were reported. Independent risk factor for E-VAP vs L-VAP was surgical diagnostic category (odds ratio [OR], 1.49 [95% confidence interval, 1.07-2.07]), whereas independent risk factors for L-VAP vs E-VAP were older age (OR, 1.01 [1.01-1.02]), high Simplified Acute Physiology Score II (OR, 1.01 [1.00-1.02]), infection on admission (OR=2.22 [1.61-3.03]), another nosocomial infection before VAP (OR, 5.88 [3.33-11.11]), and exposure to central venous catheter before VAP (OR, 4.76 [1.04-20.00]). CONCLUSIONS: E-VAP and L-VAP have different risk factors, highlighting the need for developing specific preventive measures.

Does the prognosis of cardiac arrest differ in trauma patients?

OBJECTIVE: It is proposed to not resuscitate trauma patients who have a cardiac arrest outside the hospital because they are assumed to have a dismal prognosis. Our aim was to compare the outcome of patients with traumatic or nontraumatic ("medical") out-of-hospital cardiac arrest. DESIGN: Cohort analysis of patients with out-of-hospital cardiac arrest included in the European Epinephrine Study Group's trial comparing high vs. standard doses of epinephrine. SETTING: Nine French university hospitals. PATIENTS: A total of 2,910 patients. INTERVENTIONS: Patients were successively and randomly assigned to receive repeated high doses (5 mg each) or standard doses (1 mg each) of epinephrine at 3-min intervals. MEASUREMENTS AND MAIN RESULTS: Return of spontaneous circulation, survival to hospital admission and discharge, and secondary outcome measures of 1-yr survival and neurologic outcome were recorded. In the trauma group, patients were younger (42 +/- 17 vs. 62 +/- 17 yrs, p < .001), presented with fewer witnessed out-of-hospital cardiac arrests (62.3% vs. 79.7%), and had fewer instances of ventricular fibrillation as the first documented pulseless rhythm (3.4% [95% confidence interval, 1.2-5.5%] vs. 17.3% [15.8-18.7%]). A return of spontaneous circulation was observed in 91 of 268 trauma patients (34.0% [28.3-39.6%]) compared with 797 of 2,642 medical patients (30.2% [28.4-31.9%]), and more trauma patients survived to be admitted to the hospital (29.9% [24.4-35.3%] vs. 23.5% [22.0-25.2%]). However, there was no significant difference between trauma and medical groups at hospital discharge (2.2% [0.5-4.0%] vs. 2.8% [2.1-3.4%]) and 1-yr survival (1.9% [0.3-3.5%] vs. 2.5% [1.9-3.1%]). Among patients who were discharged, a good neurologic status was observed in two trauma patients (33.3% [4.3-77.7%]) and 37 medical patients (50% [38.1-61.9%]). CONCLUSIONS: The survival and neurologic outcome of out-of-hospital cardiac arrest were not different between trauma and medical patients. This result suggests that, under the supervision of senior physicians, active resuscitation after out-of-hospital cardiac arrest is as important in trauma as in medical patients.

[The electroencephalogram is not an adequate test to confirm the diagnosis of brain death]. / L'électroencéphalogramme n'est pas un examen légitime pour la confirmation du diagnostic de mort cérébrale.

PURPOSE: In France, legislation mandates that the clinical diagnosis of brain death be confirmed by one paraclinical test before organ donation is allowed. That test may be either the electroencephalogram (EEG) or cerebral angiography. We report a case in which the clinical diagnosis of brain death was first confirmed by two EEGs performed according to the French guidelines, but ruled out by cerebral angiography. Considering that the EEG is no longer recommended to establish the diagnosis of brain death, we discuss the relevance of maintaining the EEG for brain death diagnosis in France. CLINICAL FINDINGS: A 58 yr-old man was admitted to the intensive care unit because of coma secondary to a massive subarachnoid hemorrhage with herniation below the falx shown by computed tomography. Clinical criteria of brain death were rapidly present. Two EEGs first confirmed the diagnosis but a four-vessel cerebral angiography was finally performed because the patient moved spontaneously. This cerebral angiography showed flow in the right internal carotid artery. A computed tomography performed the next day definitely confirmed the absence of brain death and organ donation did not occur. CONCLUSIONS: This case demonstrates the limitations of the EEG for this indication and suggests that angiography should be preferred. French legislation is probably maladjusted and would benefit by incorporating guidelines of other countries like Canada. International harmonization of criteria for brain death diagnosis would also be welcome.

Effects of bupivacaine, levobupivacaine and ropivacaine on myocardial relaxation.

PURPOSE: Ropivacaine and levobupivacaine were developed to reduce the risk of occasional toxicity reported with bupivacaine. While the effects of long-acting local anesthetics (LAAs) on myocardial contractility (inotropy) are well described, their effects on relaxation (lusitropy) remain largely unknown. The present study aimed to compare the effects of LAAs on rat myocardium. METHODS: Left ventricular papillary muscles of male Wistar rats were used to compare the inotropic and lusitropic responses of increasing concentrations of LAAs (10(-8) to 10(-3) M) under isometric and isotonic conditions. Data are mean % (SD) of baseline value. RESULTS: Long-acting local anesthetics induced a significant impairment of relaxation in isotonic and isometric conditions. As compared to ropivacaine, bupivacaine and levobupivacaine induced greater negative lusitropic effects in isotony [at 10(-3) M, maximum unloaded shortening velocity ((max)Vr) = 27 +/- 11 vs 13 +/- 6 and 8 +/- 5%] and isometry (at 10(-3) M, time-to-half-relaxation: 106 +/- 10 vs 127 +/- 17 and 133 +/- 17%). When the comparison was made with equipotent concentrations, the negative lusitropic effects induced with levobupivacaine were significantly greater than those of bupivacaine and ropivacaine in isometric and isotonic conditions (at 10(-3) M, (max)Vr = 7 +/- 4 vs 13 +/- 6 and 17 +/- 4 %). As previously described, LAAs also induced concentration-dependent negative inotropic effects that were greater for levobupivacaine compared to equivalent or equipotent concentrations of bupivacaine and ropivacaine. CONCLUSIONS: Long-acting local anesthetics induce marked negative inotropic and lusitropic effects. Among LAAs, levobupivacaine exerts the greater depressant effects. Impairment of calcium handling and sarcoplasmic reticulum could explain the differential responses to local anesthetics.

Methemoglobinemia by cerium nitrate poisoning.

Cerium nitrate is a topical antiseptic used with silver sulfadiazine (Flammacerium) for the treatment of serious burns. This topical agent can induce methemoglobinemia, but no cases have been reported in the recent literature. In this article, we present the case of a 16-year old girl, with third-degree burns over 95% of her body. After daily dressings of Flammacerium, on the sixth day she developed a bluish skin coloring. When tested for methemoglobinemia, levels of 31.8% were found. These returned to normal after classic treatment with Methylene blue.

Risk factors for piperacillin/tazobactam-resistant Escherichia coli in ICU patients: a clinical study.

OBJECTIVE: To determine risk factors of infections with piperacillin/tazobactam-resistant Escherichia coli in critical care patients. DESIGN: Prospective, consecutive sample survey study. SETTING: Surgical intensive care unit (ICU) in a university hospital. PATIENTS: A consecutive series of 133 patients from whom culture results were positive for E. coli during their ICU stay. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Multivariate logistic regression analysis identified the following significant independent factors associated with the emergence of a piperacillin/tazobactam-resistant Escherichia coli: prior use of amoxicillin (odds ratio, 4.15) and amoxicillin/clavulanate (odds ratio, 3.25). CONCLUSIONS: Treatment with amoxicillin or amoxicillin/clavulanate is a major risk factor for the detection of piperacillin/tazobactam-resistant E. coli in ICU patients.