Low-Avidity CD4+ T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19.

CD4+ T cells reactive against SARS-CoV-2 can be found in unexposed individuals, and these are suggested to arise in response to common cold coronavirus (CCCoV) infection. Here, we utilized SARS-CoV-2-reactive CD4+ T cell enrichment to examine the antigen avidity and clonality of these cells, as well as the relative contribution of CCCoV cross-reactivity. SARS-CoV-2-reactive CD4+ memory T cells were present in virtually all unexposed individuals examined, displaying low functional avidity and multiple, highly variable cross-reactivities that were not restricted to CCCoVs. SARS-CoV-2-reactive CD4+ T cells from COVID-19 patients lacked cross-reactivity to CCCoVs, irrespective of strong memory T cell responses against CCCoV in all donors analyzed. In severe but not mild COVID-19, SARS-CoV-2-specific T cells displayed low functional avidity and clonality, despite increased frequencies. Our findings identify low-avidity CD4+ T cell responses as a hallmark of severe COVID-19 and argue against a protective role for CCCoV-reactive T cells in SARS-CoV-2 infection.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Mutations at the conserved N-Terminal of the human Rhinovirus capsid gene VP4, and their impact on the immune response.

Rhinoviruses (RV) are the major cause of chronic obstructive pulmonary disease and are associated with exacerbation development as well as community-acquired pneumonia in children, leading to substantial morbidity, mortality, and hospital admission. Here we have examined how changes at the amino terminal of the conserved VP4 epitope of different RV serotypes may affect pulmonary cytokine and chemokine responses and disease severity. Samples positive for rhinovirus were used for genetic characterization, followed by profiling gene expression of pulmonary Th1 and Th2 cytokines/chemokines by RT-PCR arrays. Genetic sequencing and homology 3D modeling revealed changes at the amino terminal of the conserved viral protein 4 (VP4) epitope in the RV-A101 serotype, especially serine at several positions that are important for interactive binding with the host immune cells. We found dysregulation of pulmonary gene expression of Th1- and Th2-related cytokines and chemokines in RV-A 101 and RV-C 8 pneumonia patients. These findings might contribute to a better understanding of RV immunity and the potential mechanisms underlying the pathogenesis of severe RV infections, but further functional studies are needed to confirm the causal relationship.

Phenotypic comparison of human alveolar macrophages before and after in vivo rhinovirus 16 challenge.

We used mass cytometry to extensively characterize bronchoalveolar lavage macrophages before and two days after in vivo rhinovirus 16 infection in a heterogeneous population of healthy and asthma/COPD subjects. Multivariate partial least squares discriminant analysis revealed distinct clusters of alveolar macrophages before versus after the virus, suggesting changes in overall phenotype.

A Critical Role for the CXCL3/CXCL5/CXCR2 Neutrophilic Chemotactic Axis in the Regulation of Type 2 Responses in a Model of Rhinoviral-Induced Asthma Exacerbation.

Rhinovirus (RV) infections in asthmatic patients are often associated with asthma exacerbation, characterized by worsened airways hyperreactivity and increased immune cell infiltration to the airways. The C-X-C chemokines, CXCL3 and CXCL5, regulate neutrophil trafficking to the lung via CXCR2, and their expression in the asthmatic lung is associated with steroid-insensitive type 2 inflammatory signatures. Currently, the role of CXCL3 and CXCL5 in regulating neutrophilic and type 2 responses in viral-induced asthma exacerbation is unknown. Inhibition of CXCL3 or CXCL5 with silencing RNAs in a mouse model of RV-induced exacerbation of asthma attenuated the accumulation of CXCR2+ neutrophils, eosinophils, and innate lymphoid cells in the lung and decreased production of type 2 regulatory factors IL-25, IL-33, IL-5, IL-13, CCL11, and CCL24. Suppression of inflammation was associated with decreased airways hyperreactivity, mucus hypersecretion, and collagen deposition. Similar results were obtained by employing RC-3095, which has been shown to bind to CXCR2, or by depletion of neutrophils. Our data demonstrate that CXCL3 and CXCL5 may be critical in the perpetuation of RV-induced exacerbation of asthma through the recruitment of CXCR2-positive neutrophils and by promoting type 2 inflammation. Targeting the CXCL3/CXCL5/CXCR2 axis may provide a new therapeutic approach to attenuating RV-induced exacerbations of asthma.

Integrated-omics endotyping of infants with rhinovirus bronchiolitis and risk of childhood asthma.

BACKGROUND: Young children with rhinovirus (RV) infection-particularly bronchiolitis-are at high risk for developing childhood asthma. Emerging evidence suggests clinical heterogeneity within RV bronchiolitis. However, little is known about these biologically distinct subgroups (endotypes) and their relations with asthma risk. OBJECTIVE: We aimed to identify RV bronchiolitis endotypes and examine their longitudinal relations with asthma risk. METHODS: As part of a multicenter prospective cohort study of infants (age <12 months) hospitalized for bronchiolitis, we integrated clinical, RV species (RV-A, RV-B, and RV-C), nasopharyngeal microbiome (16S rRNA gene sequencing), cytokine, and metabolome (liquid chromatography tandem mass spectrometry) data collected at hospitalization. We then applied network and clustering approaches to identify bronchiolitis endotypes. We also examined their longitudinal association with risks of developing recurrent wheeze by age 3 years and asthma by age 5 years. RESULTS: Of 122 infants hospitalized for RV bronchiolitis (median age, 4 months), we identified 4 distinct endotypes-mainly characterized by RV species, microbiome, and type 2 cytokine (T2) response: endotype A, virusRV-CmicrobiomemixedT2low; endotype B, virusRV-AmicrobiomeHaemophilusT2low; endotype C, virusRSV/RVmicrobiomeStreptococcusT2low; and endotype D, virusRV-CmicrobiomeMoraxellaT2high. Compared with endotype A infants, endotype D infants had a significantly higher rate of recurrent wheeze (33% vs 64%; hazard ratio, 2.23; 95% CI, 1.00-4.96; P = .049) and a higher risk for developing asthma (28% vs 59%; odds ratio, 3.74: 95% CI, 1.21-12.6; P = .03). CONCLUSIONS: Integrated-omics analysis identified biologically meaningful RV bronchiolitis endotypes in infants, such as one characterized by RV-C infection, Moraxella-dominant microbiota, and high T2 cytokine response, at higher risk for developing recurrent wheeze and asthma. This study should facilitate further research toward validating our inferences.

Ensemble Analysis Identifies Nasal 15-Keto-PGE2 as a Predictor of Recovery in Experimental Rhinovirus Colds.

BACKGROUND: Symptom intensity during a common cold is highly variable, particularly after the illness peaks, contributing to delay in recovery. Rhinoviruses frequently cause colds and, during acute infections, generate leukotriene B4 and prostaglandin E2 (PGE2). PGE2 is known to initiate oxylipin class switching and resolution of acute inflammation. Thus, we hypothesized that during acute rhinovirus colds, oxylipins with pro-resolving capabilities reduce symptom severity and speed recovery. METHODS: Four groups of healthy volunteers were inoculated with placebo or 3 different doses of rhinovirus A16. Participants kept daily records of symptoms and contributed serial nasal lavage fluid samples. We collected semi-quantitative mass spectrometry data for 71 oxylipins in these acute samples from all participants. An ensemble analysis approach was used to further reduce this dataset. RESULTS: Levels of 15-keto-PGE2 at day 3 of the cold were consistently among the top candidates in these models of recovery symptoms. 15-keto-PGE2 was the only oxylipin with an interaction between inoculum dose and time. Acute 15-keto-PGE2 levels were inversely associated with symptoms during cold recovery in a multivariable analysis (P = .0043). CONCLUSIONS: These findings show that high 15-keto-PGE2 levels during the acute cold are associated with fewer symptoms during recovery.

IgE-mediated regulation of IL-10 and type I IFN enhances rhinovirus-induced Th2 differentiation by primary human monocytes.

Rhinovirus (RV) infections are linked to the development and exacerbation of allergic diseases including allergic asthma. IgE, another contributor to atopic disease pathogenesis, has been shown to regulate DC antiviral functions and influence T cell priming by monocytes. We previously demonstrated that IgE-mediated stimulation of monocytes alters multiple cellular functions including cytokine secretion, phagocytosis, and influenza-induced Th1 development. In this study, we investigate the effects of IgE-mediated stimulation on monocyte-driven, RV-induced T cell development utilizing primary human monocyte-T cell co-cultures. We demonstrate that IgE crosslinking of RV-exposed monocytes enhances monocyte-driven Th2 differentiation. This increase in RV-induced Th2 development was regulated by IgE-mediated inhibition of virus-induced type I IFN and induction of IL-10. These findings suggest an additional mechanism by which two clinically significant risk factors for allergic disease exacerbations-IgE-mediated stimulation and rhinovirus infection-may synergistically promote Th2 differentiation and allergic inflammation.

Toward personalization of asthma treatment according to trigger factors.

Asthma is a severe and chronic disabling disease affecting more than 300 million people worldwide. Although in the past few drugs for the treatment of asthma were available, new treatment options are currently emerging, which appear to be highly effective in certain subgroups of patients. Accordingly, there is a need for biomarkers that allow selection of patients for refined and personalized treatment strategies. Recently, serological chip tests based on microarrayed allergen molecules and peptides derived from the most common rhinovirus strains have been developed, which may discriminate 2 of the most common forms of asthma, that is, allergen- and virus-triggered asthma. In this perspective, we argue that classification of patients with asthma according to these common trigger factors may open new possibilities for personalized management of asthma.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

Genetic determinants of host immunity against human rhinovirus infections.

Human rhinoviruses (RV) are a frequent cause of respiratory tract infections with substantial morbidity and mortality in some patients. Nevertheless, the genetic basis of susceptibility to RV in humans has been relatively understudied. Experimental infections of mice and in vitro infections of human cells have indicated that various pathogen recognition receptors (TLRs, RIG-I, and MDA5) regulate innate immune responses to RV. However, deficiency of MDA5 is the only one among these so far uncovered that confers RV susceptibility in humans. Other work has shown increased RV susceptibility in patients with a polymorphism in CDHR3 that encodes the cellular receptor for RV-C entry. Here, we provide a comprehensive review of the genetic determinants of human RV susceptibility in the context of what is known about RV biology.

DUSP10 Negatively Regulates the Inflammatory Response to Rhinovirus through Interleukin-1ß Signaling.

Rhinoviral infection is a common trigger of the excessive inflammation observed during exacerbations of asthma and chronic obstructive pulmonary disease. Rhinovirus (RV) recognition by pattern recognition receptors activates the mitogen-activated protein kinase (MAPK) pathways, which are common inducers of inflammatory gene production. A family of dual-specificity phosphatases (DUSPs) can regulate MAPK function, but their roles in rhinoviral infection are not known. We hypothesized that DUSPs would negatively regulate the inflammatory response to RV infection. Our results revealed that the p38 and c-Jun N-terminal kinase (JNK) MAPKs play key roles in the inflammatory response of epithelial cells to RV infection. Three DUSPs previously shown to have roles in innate immunity (DUSPs 1, 4, and 10) were expressed in primary bronchial epithelial cells, and one of them, DUSP10, was downregulated by RV infection. Small interfering RNA-mediated knockdown of DUSP10 identified a role for the protein in negatively regulating inflammatory cytokine production in response to interleukin-1ß (IL-1ß), alone and in combination with RV, without any effect on RV replication. This study identifies DUSP10 as an important regulator of airway inflammation in respiratory viral infection.IMPORTANCE Rhinoviruses are one of the causes of the common cold. In patients with asthma or chronic obstructive pulmonary disease, viral infections, including those with rhinovirus, are the commonest cause of exacerbations. Novel therapeutics to limit viral inflammation are clearly required. The work presented here identifies DUSP10 as an important protein involved in limiting the inflammatory response in the airway without affecting immune control of the virus.

Increased type 2 inflammation post rhinovirus infection in patients with moderate asthma.

Rhinovirus (RV) infections are a major cause of exacerbations in patients with asthma. Experimental RV challenges can provide insight into the pathophysiology of viral exacerbations. Previous reports, investigating mild or moderate asthma patients, have shown an upregulation in type 2 inflammation post RV infection, however, studies specifically involving asthma patients taking inhaled corticosteroids have concentrated on symptoms and lung function, rather than the inflammatory response. Eleven moderate asthma patients were inoculated with RV. Cold symptoms and asthma control were assessed at baseline and post infection. Nasal epithelial lining fluid and bronchial alveolar lavage (BAL) fluid were collected at baseline and 4 days post infection for assessment of inflammatory proteins. Patients suffered increased cold symptoms and decreased asthma control within 7 days of infection. Antiviral mechanisms were induced following inoculation, with increases in interferon -α, ß, γ and λ, as well as CXCL10 and CXCL11. Type 2 inflammatory cytokines were also significantly elevated post RV infection in both nasal and bronchial samples. In BAL, epithelial derived IL-25 and IL-33 levels strongly correlated with Th2 cytokines, IL-4, IL-5 and IL-13. We show how experimental rhinovirus challenge regulates lung and nasal biomarkers in asthma patients taking inhaled corticosteroids. These biomarkers could be used to evaluate the effects of novel drugs for asthma.

Assessment of immunodeficiency scoring index performance in enterovirus/rhinovirus respiratory infection after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Enterovirus/rhinoviruses (EvRh) are the most common cause of respiratory virus infections in recipients of allogeneic stem cell transplantation (allo-HSCT). OBJECTIVE: We sought to analyze the value of the immunodeficiency scoring index (ISI) in predicting lower respiratory tract disease (LRTD) progression and mortality in a prospective cohort of consecutive adult (>16 years) allo-HSCT recipients with EvRh infection from December 1 2013 to December 1 2019 at two Spanish transplant centers. RESULTS: We included 234 allo-HSCT recipients with 383 EvRh episodes. Out of 383 EvRh episodes, 98 (25%) had LRTD. Multivariate logistic regression analysis identified three independent factors associated with LRTD progression: Ig G < 400 mg/dL, community-acquired respiratory virus (CARV) co-infection and high-risk ISI. Inclusion of Ig G levels and CARV co-infection in the ISI improved its performance by significantly increasing the area under the receiver operator characteristic curve (AUROC) from 0.643 to 0.734 (P = .03). Likewise, the two conditions identified by multivariate analyses as associated with higher probability of mortality were high-risk ISI and EvRh infection within 6 months after transplant. CONCLUSIONS: Our findings confirm the value of high-risk ISI in predicting both probability of EvRh LRTD and 3-month overall mortality. We also demonstrate that the original ISI could be adapted to other CARV types by including additional variables to improve its performance.

Status asthmaticus requiring extracorporeal membrane oxygenation associated with rhinovirus infection.

Objective: Evolving research links human rhinovirus (HRV) with status asthmaticus (SA) as well as severe respiratory illness in patients with atopy and asthma. This case series reviews five episodes of HRV-associated SA that required extracorporeal membrane oxygenation (ECMO). Methods: Charts of four patients, five total episodes of ECMO, with SA secondary to HRV were reviewed in this IRB-approved case series. Outcomes included demographic information, past medical history, clinical parameters and spirometry. Results: Patients (three male, one female), mean age 9 years (range 7-12 years) at the time of admission, were African American, on Medicaid, carried a diagnosis of persistent asthma, and had documented non-adherence to prescribed, daily controller medications. One patient had passive smoke exposure. All patients had a mean IgE of 734 (range 12-2497) with seasonal allergic rhinitis was diagnosed in three patients. Cases occurred in spring (3/5) and fall (2/5). Venous/venous ECMO (4/5) or venous/arterial ECMO (1/5) was continued for a mean duration of 4.2 days (range 3-7 days). Spirometry after hospitalization had a mean FEV1 of 1.59 L (81% predicted, range 69%-91%), and an FEF25%-75% 1.13 L (47.5% predicted, range 41%-65%) at an average of 16.7 weeks post ECMO. Conclusions: This case series highlights the association between persistent, poorly controlled asthma and severe SA with HRV infection resulting in ECMO. Despite life-threatening illness, these patients did not demonstrate significant large-airway obstruction following infection. However, patients showed persistently abnormal small airway function, which could be a risk factor or early evidence of vulnerability to infection.

Integrated Bioinformatics Analysis Reveals Key Candidate Genes and Cytokine Pathways Involved in COVID-19 After Rhinovirus Infection in Asthma Patients.

BACKGROUND Rhinovirus (RV) is the most common pathogen involved in asthma, and COVID-19, caused by SARS-COV-2, may be more severe in asthma patients. Here, we applied integrated bioinformatics to identify potential key genes and cytokine pathways after RV infection in asthma, and analyzed changes in angiotensin-converting enzyme 2 (ACE2), the cellular receptor of SARS-COV-2. MATERIAL AND METHODS The gene expression profile dataset GSE149273 was downloaded from NCBI-GEO, which included 90 samples of non-infected, RVA, and RVC. Differentially expressed genes (DEGs) were identified using t tests in the limma R package, and subsequently investigated by GO, KEGG, and DO analysis. Moreover, the expression of ACE2 and the proportion of immune cells were further analyzed to determine the effects of RV on cytokines. RESULTS A total of 555 DEGs of RVA and 421 of RVC were identified. There were 415 DEGs in RVA and RVC, of which 406 were upregulated and 9 were downregulated. The functional enrichment analysis showed that most DEGs were obviously enriched in cytokines, and were mainly enriched in "influenza" and "hepatitis C, chronic". In addition, the expression of ACE2 increased significantly and the proportion of immune cytokines significantly changed after RV infection. Our results suggest that RV can activate the cytokine pathway associated with COVID-19 by increasing ACE2. CONCLUSIONS The DEGs and related cytokine pathways after asthma RV infection identified using integrated bioinformatics in this study elucidate the potential link between RV and COVID-19.

Convalescent Memory T Cell Immunity in Individuals with Mild or Asymptomatic SARS-CoV-2 Infection May Result from an Evolutionarily Adapted Immune Response to Coronavirus and the 'Common Cold'.

Recent studies have shown a significant level of T cell immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in convalescent coronavirus disease 2019 (COVID-19) patients and unexposed healthy individuals. Also, SARS-CoV-2-reactive T memory cells occur in unexposed healthy individuals from endemic coronaviruses that cause the 'common cold.' The finding of the expression of adaptive SARS-CoV-2-reactive T memory cells in unexposed healthy individuals may be due to multiple cross-reactive viral protein targets following previous exposure to endemic human coronavirus infections. The opinion of the authors is that determination of protein sequence homologies across seemingly disparate viral protein libraries may provide epitope-matching data that link SARS-CoV-2-reactive T memory cell signatures to prior administration of cross-reacting vaccines to common viral pathogens. Exposure to SARS-CoV-2 initiates diverse cellular immune responses, including the associated 'cytokine storm'. Therefore, it is possible that the intact virus possesses a required degree of conformational matching, or stereoselectivity, to effectively target its receptor on multiple cell types. Therefore, conformational matching may be viewed as an evolving mechanism of viral infection and viral replication by an evolutionary modification of the angiotensin-converting enzyme 2 (ACE2) receptor required for SARS-CoV-2 binding and host cell entry. The authors propose that convalescent memory T cell immunity in individuals with mild or asymptomatic SARS-CoV-2 infection may result from an evolutionarily adapted immune response to coronavirus and the 'common cold'.

Anti-IL-5 in Mild Asthma Alters Rhinovirus-induced Macrophage, B-Cell, and Neutrophil Responses (MATERIAL). A Placebo-controlled, Double-Blind Study.

RATIONALE: Eosinophils drive pathophysiology in stable and exacerbating eosinophilic asthma, and therefore treatment is focused on the reduction of eosinophil numbers. Mepolizumab, a humanized monoclonal antibody that neutralizes IL-5 and efficiently attenuates eosinophils, proved clinically effective in severe eosinophilic asthma but not in mild asthma. OBJECTIVES: To study the effect of mepolizumab on virus-induced immune responses in mild asthma. METHODS: Patients with mild asthma, steroid-naive and randomized for eosinophil numbers, received 750 mg mepolizumab intravenously in a placebo-controlled double-blind trial, 2 weeks after which patients were challenged with rhinovirus (RV) 16. FEV1, FVC, fractional exhaled nitric oxide, symptom scores (asthma control score), viral load (PCR), eosinophil numbers, humoral (luminex, ELISA), and cellular (flow cytometry) immune parameters in blood, BAL fluid, and sputum, before and after mepolizumab and RV16, were assessed. MEASUREMENTS AND MAIN RESULTS: Mepolizumab attenuated baseline blood eosinophils and their activation, attenuated trendwise sputum eosinophils, and enhanced circulating natural killer cells. Mepolizumab did not affect FEV1, FVC, and fractional exhaled nitric oxide, neither at baseline nor after RV16. On RV16 challenge mepolizumab did not prevent eosinophil activation but did enhance local B lymphocytes and macrophages and reduce neutrophils and their activation. Mepolizumab also enhanced secretory IgA and reduced tryptase in BAL fluid. Finally, mepolizumab affected particularly RV16-induced macrophage inflammatory protein-3a, vascular endothelial growth factor-A, and IL-1RA production in BAL fluid. CONCLUSIONS: Mepolizumab failed to prevent activation of remaining eosinophils and changed RV16-induced immune responses in mild asthma. Although these latter effects likely are caused by attenuated eosinophil numbers, we cannot exclude a role for basophils. Clinical trial registered with www.clinicaltrials.gov (NCT 01520051).