Interleukin-6 inhibitory effect of natural product naringenin compared to a synthesised monoclonal antibody against life-threatening COVID-19.

Coronavirus Disease 2019 (COVID-19) has become a global pandemic in 2020 with high patient mortality due to acute respiratory distress syndrome which is possibly induced by a Cytokine release syndrome and more specifically through an interleukin-6 (IL-6) booster. Currently, IL-6/IL-6R inhibitors indicated an effective function in reducing the inflammatory markers in severe COVID-19 patients. In this comprehensively narrative review, we searched online academic databases including (Google Scholar, Web of Science, and Pub Med), the relevant literature was extracted from the databases by using search terms of COVID-19, IL-6, and IL6 inhibitor as free-text words and also with the combination with OR/AND to summarise the latest discoveries on the inhibitors of IL-6 and its receptor's especially focussing on the role of natural product, Naringin (NAR) as a flavonoid found in citrus fruits, with considerable anti-inflammatory and antiviral properties in COVID-19 treatments. Our data Therefore in comparison with other synthetic monoclonal antibodies NAR may provide a good qualification for the development of novel anti-inflammatory agents, especially against Covid 19 based on recent studies.

Evidence for an ACE2-Independent Entry Pathway That Can Protect from Neutralization by an Antibody Used for COVID-19 Therapy.

SARS-CoV-2 variants of concern (VOC) acquired mutations in the spike (S) protein, including E484K, that confer resistance to neutralizing antibodies. However, it is incompletely understood how these mutations impact viral entry into host cells. Here, we analyzed how mutations at position 484 that have been detected in COVID-19 patients impact cell entry and antibody-mediated neutralization. We report that mutation E484D markedly increased SARS-CoV-2 S-driven entry into the hepatoma cell line Huh-7 and the lung cell NCI-H1299 without augmenting ACE2 binding. Notably, mutation E484D largely rescued Huh-7 but not Vero cell entry from blockade by the neutralizing antibody Imdevimab and rendered Huh-7 cell entry ACE2-independent. These results suggest that the naturally occurring mutation E484D allows SARS-CoV-2 to employ an ACE2-independent mechanism for entry that is largely insensitive against Imdevimab, an antibody employed for COVID-19 therapy. IMPORTANCE The interaction of the SARS-CoV-2 spike protein (S) with the cellular receptor ACE2 is considered essential for infection and constitutes the key target for antibodies induced upon infection and vaccination. Here, using a surrogate system for viral entry, we provide evidence that a naturally occurring mutation can liberate SARS-CoV-2 from ACE2-dependence and that ACE2-independent entry may protect the virus from neutralization by an antibody used for COVID-19 therapy.

Preclinical discovery and development of the casirivimab + imdevimab cocktail for the treatment of novel coronavirus infection: the rise and fall.

INTRODUCTION: The ongoing COVID19 pandemic represents an unprecedented opportunity to test the feasibility of monoclonal antibody (mAb) therapies against respiratory viruses. While many hurdles were easily predictable (e.g. time to develop, scalability, and economic sustainability), mAb cocktails (i.e. the combination of two mAbs) were finally deployed in 2021, one year after the beginning of the pandemic. Of them, the REGN-COV-2 cocktail was likely the most successful experience and contributed at saving lives at the time of the wave sustained by the Delta variant of concern (VOC). AREAS COVERED: Herein, the authors review the preclinical and clinical history of the casirivimab + imdevimab cocktail for the treatment of novel coronavirus infection. The authors furthermore provide the reader with their perspectives on this cocktail including its current place in the treatment armamentarium. EXPERT OPINION: Unfortunately, results from clinical trials highlighted a very limited efficacy in inpatients; furthermore, the current evidence with regards to its lack of effectiveness against the current dominant VOC (omicron) suggests a very limited use of these drugs in the future. In the authors' opinion, this story reminds us of the limitations of mAb therapies in pandemic settings, and of the inferiority of monoclonal versus polyclonal antibody-based therapeutics in such scenarios.

Sotrovimab: First Approval.

Sotrovimab (Xevudy®) is a recombinant human monoclonal antibody targeted against the severe acute respiratory syndrome coronavirus 2. It is being developed by Vir Biotechnology in collaboration with GlaxoSmithKline for the treatment of coronavirus disease 2019 (COVID-19). Sotrovimab received its first emergency use authorization in May 2021 for the treatment of COVID-19 in the USA, with interim, emergency or conditional authorizations subsequently granted in several other countries. In December 2021, sotrovimab received its first full approval in the EU for use in adolescents (aged ≥ 12 years and weighing ≥ 40 kg) and adults with COVID-19 who do not require oxygen supplementation and who are at high risk of progressing to severe COVID-19. This article summarizes the milestones in the development of sotrovimab leading to this first approval.

Antibody escape and global spread of SARS-CoV-2 lineage A.27.

In spring 2021, an increasing number of infections was observed caused by the hitherto rarely described SARS-CoV-2 variant A.27 in south-west Germany. From December 2020 to June 2021 this lineage has been detected in 31 countries. Phylogeographic analyses of A.27 sequences obtained from national and international databases reveal a global spread of this lineage through multiple introductions from its inferred origin in Western Africa. Variant A.27 is characterized by a mutational pattern in the spike gene that includes the L18F, L452R and N501Y spike amino acid substitutions found in various variants of concern but lacks the globally dominant D614G. Neutralization assays demonstrate an escape of A.27 from convalescent and vaccine-elicited antibody-mediated immunity. Moreover, the therapeutic monoclonal antibody Bamlanivimab and partially the REGN-COV2 cocktail fail to block infection by A.27. Our data emphasize the need for continued global monitoring of novel lineages because of the independent evolution of new escape mutations.

The Omicron variant is highly resistant against antibody-mediated neutralization: Implications for control of the COVID-19 pandemic.

The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike protein raised concerns that the virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent patients or individuals vaccinated with the BioNTech-Pfizer vaccine (BNT162b2) with 12- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1 (Astra Zeneca-Oxford)/BNT162b2 vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.

Mortality Rates Among Hospitalized Patients With COVID-19 Infection Treated With Tocilizumab and Corticosteroids: A Bayesian Reanalysis of a Previous Meta-analysis.

Importance: A World Health Organization (WHO) meta-analysis found that tocilizumab was associated with reduced mortality in hospitalized patients with COVID-19. However, uncertainty remains concerning the magnitude of tocilizumab's benefits and whether its association with mortality benefit is similar across respiratory subgroups. Objective: To use bayesian methods to assess the magnitude of mortality benefit associated with tocilizumab and the differences between respiratory support subgroups in hospitalized patients with COVID-19. Design, Setting, and Participants: A bayesian hierarchical reanalysis of the WHO meta-analysis of tocilizumab studies published in 2020 and 2021 was performed. Main results were estimated using weakly informative priors to exert little influence on the observed data. The robustness of these results was evaluated using vague and informative priors. The studies featured in the meta-analysis were randomized clinical tocilizumab trials of hospitalized patients with COVID-19. Only patients receiving corticosteroids were included. Interventions: Usual care plus tocilizumab in comparison with usual care or placebo. Main Outcomes and Measures: All-cause mortality at 28 days after randomization. Results: Among the 5339 patients included in this analysis, most were men, with mean ages between 56 and 66 years. There were 2117 patients receiving simple oxygen only, 2505 receiving noninvasive ventilation (NIV), and 717 receiving invasive mechanical ventilation (IMV) in 15 studies from multiple countries and continents. Assuming weakly informative priors, the overall odds ratios (ORs) for survival were 0.70 (95% credible interval [CrI], 0.50-0.91) for patients receiving simple oxygen only, 0.81 (95% CrI, 0.63-1.03) for patients receiving NIV, and 0.89 (95% CrI, 0.61-1.22) for patients receiving IMV, respectively. The posterior probabilities of any benefit (OR <1) were notably different between patients receiving simple oxygen only (98.9%), NIV (95.5%), and IMV (75.4%). The posterior probabilities of a clinically meaningful association (absolute mortality risk difference >1%) were greater than 95% in patients receiving simple oxygen only and greater than 90% in patients receiving NIV. In contrast, the posterior probability of this clinically meaningful association was only approximately 67% in patients receiving IMV. The probabilities of tocilizumab superiority in the simple oxygen only subgroup compared with the NIV and IMV subgroups were 85% and 90%, respectively. Predictive intervals highlighted that only 72.1% of future tocilizumab IMV studies would show benefit. The conclusions did not change with different prior distributions. Conclusions and Relevance: In this bayesian reanalysis of a previous meta-analysis of 15 studies of hospitalized patients with COVID-19 treated with tocilizumab and corticosteroids, use of simple oxygen only and NIV was associated with a probability of a clinically meaningful mortality benefit from tocilizumab. Future research should clarify whether patients receiving IMV also benefit from tocilizumab.

In-Silico Analysis of Monoclonal Antibodies against SARS-CoV-2 Omicron.

Omicron was designated by the WHO as a VOC on 26 November 2021, only 4 days after its sequence was first submitted. However, the impact of Omicron on current antibodies and vaccines remains unknown and evaluations are still a few weeks away. We analysed the mutations in the Omicron variant against epitopes. In our database, 132 epitopes of the 120 antibodies are classified into five groups, namely NTD, RBD-1, RBD-2, RBD-3, and RBD-4. The Omicron mutations impact all epitopes in NTD, RBD-1, RBD-2, and RBD-3, with no antibody epitopes spared by these mutations. Only four out of 120 antibodies may confer full resistance to mutations in the Omicron spike, since all antibodies in these three groups contain one or more epitopes that are affected by these mutations. Of all antibodies under EUA, the neutralisation potential of Etesevimab, Bamlanivimab, Casirivimab, Imdevima, Cilgavimab, Tixagevimab, Sotrovimab, and Regdanvimab might be dampened to varying degrees. Our analysis suggests the impact of Omicron on current therapeutic antibodies by the Omicron spike mutations may also apply to current COVID-19 vaccines.

Etesevimab in combination with JS026 neutralizing SARS-CoV-2 and its variants.

The neutralizing antibody is a potential therapeutic for the ongoing COVID-19 pandemic. As an antiviral agent, numerous mAbs recognize the epitopes that overlap with ACE2-binding sites in the SARS-CoV-2-RBD. Some studies have shown that residual changes on the spike protein can significantly decrease the efficiency of neutralizing antibodies. To address this issue, a therapeutic cocktail could be an effective countermeasure. In the present study, we isolated a fully human neutralizing antibody, JS026, from a convalescent patient. The comparative analysis revealed that JS026 binding to SARS-CoV-2-RBD mainly located between epitopes for class 2 and class 3 mAbs as opposed to that of class 1 (etesevimab) antibodies. A cocktail of etesevimab and JS026 increased neutralizing efficacy against both wild-type SARS-CoV-2 and the recent emergence of Alpha, Beta, Gamma, and Delta variants. JS026 and the cocktail reduced virus titers in the infected lungs of hACE2 transgenic mice and relieved pathological changes. These findings would benefit antibody-based therapeutic countermeasures in the treatment of COVID-19.

Arrhythmogenic mechanisms of interleukin-6 combination with hydroxychloroquine and azithromycin in inflammatory diseases.

Inflammatory diseases including COVID-19 are associated with a cytokine storm characterized by high interleukin-6 (IL-6) titers. In particular, while recent studies examined COVID-19 associated arrhythmic risks from cardiac injury and/or from pharmacotherapy such as the combination of azithromycin (AZM) and hydroxychloroquine (HCQ), the role of IL-6 per se in increasing the arrhythmic risk remains poorly understood. The objective is to elucidate the electrophysiological basis of inflammation-associated arrhythmic risk in the presence of AZM and HCQ. IL-6, AZM and HCQ were concomitantly administered to guinea pigs in-vivo and in-vitro. Electrocardiograms, action potentials and ion-currents were analyzed. IL-6 alone or the combination AZM + HCQ induced mild to moderate reduction in heart rate, PR-interval and corrected QT (QTc) in-vivo and in-vitro. Notably, IL-6 alone was more potent than the combination of the two drugs in reducing heart rate, increasing PR-interval and QTc. In addition, the in-vivo or in-vitro combination of IL-6 + AZM + HCQ caused severe bradycardia, conduction abnormalities, QTc prolongation and asystole. These electrocardiographic abnormalities were attenuated in-vivo by tocilizumab (TCZ), a monoclonal antibody against IL-6 receptor, and are due in part to the prolongation of action potential duration and selective inhibition of Na+, Ca2+ and K+ currents. Inflammation confers greater risk for arrhythmia than the drug combination therapy. As such, in the setting of elevated IL-6 during inflammation caution must be taken when co-administering drugs known to predispose to fatal arrhythmias and TCZ could be an important player as a novel anti-arrhythmic agent. Thus, identifying inflammation as a critical culprit is essential for proper management.

Immunomodulatory agents for COVID-19 treatment: possible mechanism of action and immunopathology features.

The novel coronavirus pandemic has emerged as one of the significant medical-health challenges of the current century. The World Health Organization has named this new virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since the first detection of SARS-CoV-2 in November 2019 in Wuhan, China, physicians, researchers, and others have made it their top priority to find drugs and cures that can effectively treat patients and reduce mortality rates. The symptoms of Coronavirus Disease 2019 (COVID-19) include fever, dry cough, body aches, and anosmia. Various therapeutic compounds have been investigated and applied to mitigate the symptoms in COVID-19 patients and cure the disease. Degenerative virus analyses of the infection incidence and COVID-19 have demonstrated that SARS-CoV-2 penetrates the pulmonary alveoli's endothelial cells through Angiotensin-Converting Enzyme 2 (ACE2) receptors on the membrane, stimulates various signaling pathways and causes excessive secretion of cytokines. The continuous triggering of the innate and acquired immune system, as well as the overproduction of pro-inflammatory factors, cause a severe condition in the COVID-19 patients, which is called "cytokine storm". It can lead to acute respiratory distress syndrome (ARDS) in critical patients. Severe and critical COVID-19 cases demand oxygen therapy and mechanical ventilator support. Various drugs, including immunomodulatory and immunosuppressive agents (e.g., monoclonal antibodies (mAbs) and interleukin antagonists) have been utilized in clinical trials. However, the studies and clinical trials have documented diverging findings, which seem to be due to the differences in these drugs' possible mechanisms of action. These drugs' mechanism of action generally includes suppressing or modulating the immune system, preventing the development of cytokine storm via various signaling pathways, and enhancing the blood vessels' diameter in the lungs. In this review article, multiple medications from different drug families are discussed, and their possible mechanisms of action are also described.

Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations.

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD80 dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb's efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.

Interleukin-6 in SARS-CoV-2 induced disease: Interactions and therapeutic applications.

Interleukin-6 (IL-6) is a multi-tasking cytokine that represents high activity in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cancer. High concentration of this pleiotropic cytokine accounts for hyperinflammation and cytokine storm, and is related to multi-organ failure in patients with SARS-CoV-2 induced disease. IL-6 promotes lymphopenia and increases C-reactive protein (CRP) in such cases. However, blockade of IL-6 is not a full-proof of complete response. Hypoxia, hypoxemia, aberrant angiogenesis and chronic inflammation are inter-related events occurring as a response to the SARS-CoV-2 stimulatory effect on high IL-6 activity. Taking both pro- and anti-inflammatory activities will make complex targeting IL-6 in patient with SARS-CoV-2 induced disease. The aim of this review was to discuss about interactions occurring within the body of patients with SARS-CoV-2 induced disease who are representing high IL-6 levels, and to determine whether IL-6 inhibition therapy is effective for such patients or not. We also address the interactions and targeted therapies in cancer patients who also have SARS-CoV-2 induced disease.

Chronic pharmacological antagonism of the GM-CSF receptor in mice does not replicate the pulmonary alveolar proteinosis phenotype but does alter lung surfactant turnover.

Granulocyte macrophage colony stimulating factor (GM-CSF) is a key participant in, and a clinical target for, the treatment of inflammatory diseases including rheumatoid arthritis (RA). Therapeutic inhibition of GM-CSF signalling using monoclonal antibodies to the α-subunit of the GM-CSF receptor (GMCSFRα) has shown clear benefit in patients with RA, giant cell arteritis (GCAs) and some efficacy in severe SARS-CoV-2 infection. However, GM-CSF autoantibodies are associated with the development of pulmonary alveolar proteinosis (PAP), a rare lung disease characterised by alveolar macrophage (AM) dysfunction and the accumulation of surfactant lipids. We assessed how the anti-GMCSFRα approach might impact surfactant turnover in the airway. Female C57BL/6J mice received a mouse-GMCSFRα blocking antibody (CAM-3003) twice per week for up to 24 weeks. A parallel, comparator cohort of the mouse PAP model, GM-CSF receptor ß subunit (GMCSFRß) knock-out (KO), was maintained up to 16 weeks. We assessed lung tissue histopathology alongside lung phosphatidylcholine (PC) metabolism using stable isotope lipidomics. GMCSFRß KO mice reproduced the histopathological and biochemical features of PAP, accumulating surfactant PC in both broncho-alveolar lavage fluid (BALF) and lavaged lung tissue. The incorporation pattern of methyl-D9-choline showed impaired catabolism and not enhanced synthesis. In contrast, chronic supra-pharmacological CAM-3003 exposure (100 mg/kg) over 24 weeks did not elicit a histopathological PAP phenotype despite some changes in lung PC catabolism. Lack of significant impairment of AM catabolic function supports clinical observations that therapeutic antibodies to this pathway have not been associated with PAP in clinical trials.

High Induction of IL-6 Secretion From hUCMSCs Optimize the Potential of hUCMSCs and TCZ as Therapy for COVID-19-Related ARDS.

Biological and cellular interleukin-6 (IL-6)-related therapies have been used to treat severe COVID-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure, which prompted further exploration of the role of IL-6 in human umbilical cord mesenchymal stem cell (hUCMSC) therapy. Peripheral blood mononuclear cells (PBMCs) were responders cocultured with hUCMSCs or exogenous IL-6. A PBMC suppression assay was used to analyze the anti-inflammatory effects via MTT assay. The IL-6 concentration in the supernatant was measured using ELISA. The correlation between the anti-inflammatory effect of hUCMSCs and IL-6 levels and the relevant roles of IL-6 and IL-6 mRNA expression was analyzed using the MetaCore functional network constructed from gene microarray data. The location of IL-6 and IL-6 receptor (IL-6R) expression was further evaluated. We reported that hUCMSCs did not initially exert any inhibitory effect on PHA-stimulated proliferation; however, a potent inhibitory effect on PHA-stimulated proliferation was observed, and the IL-6 concentration reached approximately 1000 ng/mL after 72 hours. Exogenous 1000 ng/mL IL-6 inhibited PHA-stimulated inflammation but less so than hUCMSCs. The inhibitory effects of hUCMSCs on PHA-stimulated PBMCs disappeared after adding an IL-6 neutralizing antibody or pretreatment with tocilizumab (TCZ), an IL-6R antagonist. hUCMSCs exert excellent anti-inflammatory effects by inducing higher IL-6 levels, which is different from TCZ. High concentration of IL-6 cytokine secretion plays an important role in the anti-inflammatory effect of hUCMSC therapy. Initial hUCMSC therapy, followed by TCZ, seems to optimize the therapeutic potential to treat COVID-19-related acute respiratory distress syndrome (ARDS).

Effect of tezepelumab on airway inflammatory cells, remodelling, and hyperresponsiveness in patients with moderate-to-severe uncontrolled asthma (CASCADE): a double-blind, randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine. In phase 2b and 3 studies, tezepelumab significantly reduced exacerbations versus placebo in patients with severe uncontrolled asthma, irrespective of baseline levels of type 2 inflammatory biomarkers. We investigated the mechanism of action of tezepelumab by assessing its effects on airway inflammatory cells, airway remodelling, and airway hyperresponsiveness. METHODS: CASCADE was an exploratory, double-blind, randomised, placebo-controlled, parallel-group, phase 2 study done in 27 medical centres in Canada, Denmark, Germany, the UK, and the USA. Adults aged 18-75 years with uncontrolled, moderate-to-severe asthma were randomly assigned (1:1) to receive tezepelumab 210 mg or placebo administered subcutaneously every 4 weeks for a planned 28 weeks, extended to up to 52 weeks if COVID-19-related disruption delayed participants' end-of-treatment assessments. Randomisation was balanced and stratified by blood eosinophil count. The primary endpoint was the change from baseline to the end of treatment in the number of airway submucosal inflammatory cells in bronchoscopic biopsy samples. Eosinophils, neutrophils, CD3+ T cells, CD4+ T cells, tryptase+ mast cells, and chymase+ mast cells were evaluated separately. This endpoint was also assessed in subgroups according to baseline type 2 inflammatory biomarker levels, including blood eosinophil count. Airway remodelling was assessed via the secondary endpoints of change from baseline in reticular basement membrane thickness and epithelial integrity (proportions of denuded, damaged, and intact epithelium). Exploratory outcomes included airway hyperresponsiveness to mannitol. All participants who completed at least 20 weeks of study treatment, had an end-of-treatment visit up to 8 weeks after the last dose of study drug, and had evaluable baseline and end-of-treatment bronchoscopies were included in the primary efficacy analysis. All participants who received at least one dose of study drug were included in the safety analyses. This study is registered with ClinicalTrials.gov, NCT03688074. FINDINGS: Between Nov 2, 2018, and Nov 16, 2020, 250 patients were enrolled, 116 of whom were randomly assigned (59 to tezepelumab, 57 to placebo). 48 in the tezepelumab group and 51 in the placebo group completed the study and were assessed for the primary endpoint. Treatment with tezepelumab resulted in a nominally significantly greater reduction from baseline to the end of treatment in airway submucosal eosinophils versus placebo (ratio of geometric least-squares means 0·15 [95% CI 0·05-0·41]; nominal p<0·0010), with the difference seen across all baseline biomarker subgroups. There were no significant differences between treatment groups in the other cell types evaluated (ratio of geometric least-squares means: neutrophils 1·36 [95% CI 0·94-1·97]; CD3+ T cells 1·12 [0·86-1·46]; CD4+ T cells 1·18 [0·90-1·55]; tryptase+ mast cells 0·83 [0·61-1·15]; chymase+ mast cells 1·19 [0·67-2·10]; all p>0·10). In assessment of secondary endpoints, there were no significant differences between treatment groups in reticular basement membrane thickness and epithelial integrity. In an exploratory analysis, the reduction in airway hyperresponsiveness to mannitol was significantly greater with tezepelumab versus placebo (least-squares mean change from baseline in interpolated or extrapolated provoking dose of mannitol required to induce ≥15% reduction in FEV1 from baseline: tezepelumab 197·4 mg [95% CI 107·9 to 286·9]; placebo 58·6 mg [-30·1 to 147·33]; difference 138·8 [14·2 to 263·3], nominal p=0·030). Adverse events were reported in 53 (90%) patients in the tezepelumab group and 51 (90%) patients in the placebo group, and there were no safety findings of concern. INTERPRETATION: The improvements in asthma clinical outcomes observed in previous studies with tezepelumab are probably driven, at least in part, by reductions in eosinophilic airway inflammation, as shown here by reduced airway eosinophil counts regardless of baseline blood eosinophil count. Tezepelumab also reduced airway hyperresponsiveness to mannitol, indicating that TSLP blockade might have additional benefits in asthma beyond reducing type 2 airway inflammation. FUNDING: AstraZeneca and Amgen.