Mammalian Meat Allergy and IgE to Alpha-gal in in Central Virginia: findings from a COVID-19 vaccine and patient cohort.

BACKGROUND: IgE to galactose-alpha-1,3-galactose (alpha-gal) is linked with tick bites and an important cause of anaphylaxis and urticarial reactions to mammalian meat. The "alpha-gal syndrome" (AGS) is recognized as being common in the southeastern USA, however prevalence studies are lacking and open questions remain about risk factors and clinical presentation of alpha-gal sensitization. OBJECTIVE: Here we characterized the prevalence, as well as presentation and risk factors, of AGS and alpha-gal IgE sensitization in adults in central Virginia recruited without regards to history of allergic disease. METHODS: Adults in central Virginia, primarily University of Virginia Health employees, were recruited as part of a COVID-19 vaccine study. Subjects provided at least one blood sample and answered questionnaires about medical and dietary history. ImmunoCAP was used for IgE assays and ABO blood group was assessed by reverse typing using stored serum. Biobanked serum from COVID-19 patients was also investigated. RESULTS: Of 267 enrollees, median age was 42, 76% were female and 43 (16%) were sensitized to alpha-gal (cut-off 0.1 IU/mL), of which mammalian meat allergy was reported by 7 (2.6%). Sensitized subjects were i) older, ii) had higher total IgE levels but similar frequency of IgE to common respiratory allergens, and iii) were more likely to report tick bites than non-sensitized subjects. Among those who were sensitized, alpha-gal IgE levels were higher among meat allergic than non-allergic subjects (GM 9.0 vs 0.5 IU/mL, P<0.001). Mammalian meat and dairy consumption was common in individuals with low-level sensitization. CONCLUSION: In central Virginia AGS is a dominant cause of adult food allergy with a prevalence approaching or exceeding 2%.

Distinct Type 1 Immune Networks Underlie the Severity of Restrictive Lung Disease after COVID-19.

The variable etiology of persistent breathlessness after COVID-19 have confounded efforts to decipher the immunopathology of lung sequelae. Here, we analyzed hundreds of cellular and molecular features in the context of discrete pulmonary phenotypes to define the systemic immune landscape of post-COVID lung disease. Cluster analysis of lung physiology measures highlighted two phenotypes of restrictive lung disease that differed by their impaired diffusion and severity of fibrosis. Machine learning revealed marked CCR5+CD95+ CD8+ T-cell perturbations in mild-to-moderate lung disease, but attenuated T-cell responses hallmarked by elevated CXCL13 in more severe disease. Distinct sets of cells, mediators, and autoantibodies distinguished each restrictive phenotype, and differed from those of patients without significant lung involvement. These differences were reflected in divergent T-cell-based type 1 networks according to severity of lung disease. Our findings, which provide an immunological basis for active lung injury versus advanced disease after COVID-19, might offer new targets for treatment.

ACE-2-like enzymatic activity is associated with immunoglobulin in COVID-19 patients.

Many mechanisms responsible for COVID-19 pathogenesis are well-established, but COVID-19 includes features with unclear pathogenesis, such as autonomic dysregulation, coagulopathies, and high levels of inflammation. The receptor for the SARS-CoV-2 spike protein receptor-binding domain (RBD) is angiotensin-converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop antibodies that have a negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity-ACE2-like abzymes. To explore this hypothesis, we studied patients hospitalized with COVID-19 who had plasma samples available obtained about 7 days after admission. ACE2 is a metalloprotease that requires Zn2+ for activity. However, we found that the plasma from some patients studied could specifically cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using disodium EDTA anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+ was added or the plasma was diluted to decrease EDTA concentration. After processing samples by 100 kDa size exclusion columns and protein A/G adsorption, which depleted immunoglobulin by >99.99%, the plasma samples did not cleave the ACE2 substrate peptide. The data suggest that some patients with COVID-19 develop antibodies with abzyme-like activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate many physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure COVID-19 pathogenesis. IMPORTANCE: We provide what we believe to be the first description of angiotensin-converting enzyme 2 (ACE2)-like enzymatic activity associated with immunoglobulin in COVID-19 patients. COVID-19 includes many puzzling clinical features that have unclear pathogenesis, including a hyperinflammatory state, abnormalities of the clotting cascade, and blood pressure instability. We hypothesized that some patients with COVID-19 patients may produce antibodies against SARS-CoV-2 with enzymatic activity, or abzymes, that target important proteolytic regulatory cascades. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein binds ACE2 on the surface of the future host cell. This means that the RBD has a negative molecular image of ACE2. We hypothesized that some antibodies produced against the RBD would have, in turn, a negative molecular image of the RBD sufficiently similar to ACE2 to have ACE2-like catalytic activity. In other words, some anti-RBD antibodies would be ACE2-like abzymes. Abzymes elicited by SARS-CoV-2 infection have the potential to affect host physiology.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19.

The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1ß, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1ß after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

Proximal immune-epithelial progenitor interactions drive chronic tissue sequelae post COVID-19.

The long-term physiological consequences of SARS-CoV-2, termed Post-Acute Sequelae of COVID-19 (PASC), are rapidly evolving into a major public health concern. The underlying cellular and molecular etiology remain poorly defined but growing evidence links PASC to abnormal immune responses and/or poor organ recovery post-infection. Yet, the precise mechanisms driving non-resolving inflammation and impaired tissue repair in the context of PASC remain unclear. With insights from three independent clinical cohorts of PASC patients with abnormal lung function and/or viral infection-mediated pulmonary fibrosis, we established a clinically relevant mouse model of post-viral lung sequelae to investigate the pathophysiology of respiratory PASC. By employing a combination of spatial transcriptomics and imaging, we identified dysregulated proximal interactions between immune cells and epithelial progenitors unique to the fibroproliferation in respiratory PASC but not acute COVID-19 or idiopathic pulmonary fibrosis (IPF). Specifically, we found a central role for lung-resident CD8+ T cell-macrophage interactions in maintaining Krt8hi transitional and ectopic Krt5+ basal cell progenitors, thus impairing alveolar regeneration and driving fibrotic sequelae after acute viral pneumonia. Mechanistically, CD8+ T cell derived IFN-γ and TNF stimulated lung macrophages to chronically release IL-1ß, resulting in the abnormal accumulation of dysplastic epithelial progenitors and fibrosis. Notably, therapeutic neutralization of IFN-γ and TNF, or IL-1ß after the resolution of acute infection resulted in markedly improved alveolar regeneration and restoration of pulmonary function. Together, our findings implicate a dysregulated immune-epithelial progenitor niche in driving respiratory PASC. Moreover, in contrast to other approaches requiring early intervention, we highlight therapeutic strategies to rescue fibrotic disease in the aftermath of respiratory viral infections, addressing the current unmet need in the clinical management of PASC and post-viral disease.

High-dimensional comparison of monocytes and T cells in post-COVID and idiopathic pulmonary fibrosis.

Introduction: Up to 30% of hospitalized COVID-19 patients experience persistent sequelae, including pulmonary fibrosis (PF). Methods: We examined COVID-19 survivors with impaired lung function and imaging worrisome for developing PF and found within six months, symptoms, restriction and PF improved in some (Early-Resolving COVID-PF), but persisted in others (Late-Resolving COVID-PF). To evaluate immune mechanisms associated with recovery versus persistent PF, we performed single-cell RNA-sequencing and multiplex immunostaining on peripheral blood mononuclear cells from patients with Early- and Late-Resolving COVID-PF and compared them to age-matched controls without respiratory disease. Results and discussion: Our analysis showed circulating monocytes were significantly reduced in Late-Resolving COVID-PF patients compared to Early-Resolving COVID-PF and non-diseased controls. Monocyte abundance correlated with pulmonary function forced vital capacity and diffusion capacity. Differential expression analysis revealed MHC-II class molecules were upregulated on the CD8 T cells of Late-Resolving COVID-PF patients but downregulated in monocytes. To determine whether these immune signatures resembled other interstitial lung diseases, we analyzed samples from Idiopathic Pulmonary Fibrosis (IPF) patients. IPF patients had a similar marked decrease in monocyte HLA-DR protein expression compared to Late-Resolving COVID-PF patients. Our findings indicate decreased circulating monocytes are associated with decreased lung function and uniquely distinguish Late-Resolving COVID-PF from Early-Resolving COVID-PF, IPF, and non-diseased controls.

Epstein-Barr Virus and Human Herpesvirus-6 Reactivation in Acute COVID-19 Patients.

Beyond their pulmonary disease, many COVID-19 patients experience a complex constellation of characteristics, including hyperinflammatory responses, autoimmune disorders, and coagulopathies. However, the pathogenesis of these aspects of COVID-19 is obscure. More than 90% of people are latently infected with the lymphotropic herpesviruses Epstein-Barr Virus (EBV) and/or Human Herpesvirus-6 (HHV-6). Some of the inflammatory features of COVID-19 resemble clinical syndromes seen during EBV and HHV-6 infection, and these latent viruses can be reactivated by inflammatory mediators. We hypothesized that EBV and HHV-6 reactivation might be a common feature of early COVID-19, particularly in patients with more inflammation. We tested for EBV and HHV-6 reactivation in 67 patients acutely hospitalized with COVID-19 using previously validated quantitative PCR assays on the plasma. In our cohort, we found that 15/67 (22.4%) patients had detectable EBV and 3/67 (4.5%) had detectable HHV-6. This frequency of activation is somewhat more than the frequency reported for some healthy cohorts, such as blood donors and other healthy control cohorts. There was no association between EBV or HHV-6 and markers indicative of more inflammatory disease. We conclude that EBV and HHV-6 activation at about day 7 of hospitalization occurred in a modest fraction of our cohort of COVID-19 patients and was not associated with high levels of inflammation. In the modest fraction of patients, EBV and HHV-6 reactivation could contribute to some features of acute disease and pre-disposition to post-acute sequelae in a subset of patients.

Cluster analysis of nasal cytokines during rhinovirus infection identifies different immunophenotypes in both children and adults with allergic asthma.

BACKGROUND: Infection with rhinovirus (RV) is a major risk factor for disease exacerbations in patients with allergic asthma. This study analysed a broad set of cytokines in the noses of children and adults with asthma during RV infection in order to identify immunophenotypes that may link to virus-induced episodes. METHODS: Nasal wash specimens were analysed in children (n = 279 [healthy, n = 125; stable asthma, n = 64; wheeze, n = 90], ages 2-12) who presented to a hospital emergency department, and in adults (n = 44 [healthy, n = 13; asthma, n = 31], ages 18-38) who were experimentally infected with RV, including a subset who received anti-IgE. Cytokines were measured by multiplex bead assay and data analysed by univariate and multivariate methods to test relationships to viral load, allergic status, airway inflammation, and clinical outcomes. RESULTS: Analysis of a core set of 7 cytokines (IL-6, CXCL8/IL-8, IL-15, EGF, G-CSF, CXCL10/IP-10 and CCL22/MDC) revealed higher levels in children with acute wheeze versus those with stable asthma or controls. Multivariate analysis identified two clusters that were enriched for acutely wheezing children; one displaying high viral load ("RV-high") with robust secretion of CXCL10, and the other displaying high IgE with elevated EGF, CXCL8 and both eosinophil- and neutrophil-derived mediators. Broader assessment of 39 cytokines confirmed that children with acute wheeze were not deficient in type 1 anti-viral responses. Analysis of 18 nasal cytokines in adults with asthma who received RV challenge identified two clusters; one that was "RV-high" and linked to robust induction of anti-viral cytokines and anti-IgE; and the other associated with more severe symptoms and a higher inflammatory state featuring eosinophil and neutrophil factors. CONCLUSIONS: The results confirm the presence of different immunophenotypes linked to parameters of airway disease in both children and adults with asthma who are infected with RV. Such discrepancies may reflect the ability to regulate anti-viral responses.

Trajectory of IgG to SARS-CoV-2 After Vaccination With BNT162b2 or mRNA-1273 in an Employee Cohort and Comparison With Natural Infection.

Three COVID-19 vaccines have received FDA-authorization and are in use in the United States, but there is limited head-to-head data on the durability of the immune response elicited by these vaccines. Using a quantitative assay we studied binding IgG antibodies elicited by BNT162b2, mRNA-1273 or Ad26.COV2.S in an employee cohort over a span out to 10 months. Age and sex were explored as response modifiers. Of 234 subjects in the vaccine cohort, 114 received BNT162b2, 114 received mRNA-1273 and six received Ad26.COV2.S. IgG levels measured between seven to 20 days after the second vaccination were similar in recipients of BNT162b2 and mRNA-127 and were ~50-fold higher than in recipients of Ad26.COV2.S. However, by day 21 and at later time points IgG levels elicited by BNT162b2 were lower than mRNA-1273. Accordingly, the IgG decay curve was steeper for BNT162b2 than mRNA-1273. Age was a significant modifier of IgG levels in recipients of BNT162b2, but not mRNA-1273. After six months, IgG levels elicited by BNT162b2, but not mRNA-1273, were lower than IgG levels in patients who had been hospitalized with COVID-19 six months earlier. Similar findings were observed when comparing vaccine-elicited antibodies with steady-state IgG targeting seasonal human coronaviruses. Differential IgG decay could contribute to differences observed in clinical protection over time between BNT162b2 and mRNA-1273.

Unsupervised machine learning reveals key immune cell subsets in COVID-19, rhinovirus infection, and cancer therapy.

For an emerging disease like COVID-19, systems immunology tools may quickly identify and quantitatively characterize cells associated with disease progression or clinical response. With repeated sampling, immune monitoring creates a real-time portrait of the cells reacting to a novel virus before disease-specific knowledge and tools are established. However, single cell analysis tools can struggle to reveal rare cells that are under 0.1% of the population. Here, the machine learning workflow Tracking Responders EXpanding (T-REX) was created to identify changes in both rare and common cells across human immune monitoring settings. T-REX identified cells with highly similar phenotypes that localized to hotspots of significant change during rhinovirus and SARS-CoV-2 infections. Specialized MHCII tetramer reagents that mark rhinovirus-specific CD4+ cells were left out during analysis and then used to test whether T-REX identified biologically significant cells. T-REX identified rhinovirus-specific CD4+ T cells based on phenotypically homogeneous cells expanding by ≥95% following infection. T-REX successfully identified hotspots of virus-specific T cells by comparing infection (day 7) to either pre-infection (day 0) or post-infection (day 28) samples. Plotting the direction and degree of change for each individual donor provided a useful summary view and revealed patterns of immune system behavior across immune monitoring settings. For example, the magnitude and direction of change in some COVID-19 patients was comparable to blast crisis acute myeloid leukemia patients undergoing a complete response to chemotherapy. Other COVID-19 patients instead displayed an immune trajectory like that seen in rhinovirus infection or checkpoint inhibitor therapy for melanoma. The T-REX algorithm thus rapidly identifies and characterizes mechanistically significant cells and places emerging diseases into a systems immunology context for comparison to well-studied immune changes.

Quantitative Measurement of IgG to Severe Acute Respiratory Syndrome Coronavirus-2 Proteins Using ImmunoCAP.

BACKGROUND: Detailed understanding of the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV)-2, the cause of coronavirus disease 2019 (CO-VID-19) has been hampered by a lack of quantitative antibody assays. OBJECTIVE: The objective was to develop a quantitative assay for IgG to SARS-CoV-2 proteins that could be implemented in clinical and research laboratories. METHODS: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding domain (RBD) or nucleocapsid protein to the solid phase of the ImmunoCAP. Plasma and serum samples from patients hospitalized with COVID-19 (n = 60) and samples from donors banked before the emergence of COVID-19 (n = 109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform. RESULTS: At a cutoff of 2.5 µg/mL, the assay demonstrated sensitivity and specificity exceeding 95% for IgG to both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 µg/mL (IQR 18-52) and 24.5 µg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples, there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 µg/mL, or 1% of total IgG. CONCLUSIONS: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to mutated COVID-19 proteins, has good performance characteristics, and has a readout in standardized units.

Quantitative measurement of IgG to SARS-CoV-2 proteins using ImmunoCAP.

BACKGROUND: Detailed understanding of the immune response to SARS-CoV-2, the cause of coronavirus disease 2019 (COVID-19), has been hampered by a lack of quantitative antibody assays. OBJECTIVE: To develop a quantitative assay for IgG to SARS-CoV-2 proteins that could readily be implemented in clinical and research laboratories. METHODS: The biotin-streptavidin technique was used to conjugate SARS-CoV-2 spike receptor-binding-domain (RBD) or nucleocapsid protein to the solid-phase of the ImmunoCAP resin. Plasma and serum samples from patients with COVID-19 (n=51) and samples from donors banked prior to the emergence of COVID-19 (n=109) were used in the assay. SARS-CoV-2 IgG levels were followed longitudinally in a subset of samples and were related to total IgG and IgG to reference antigens using an ImmunoCAP 250 platform. RESULTS: Performance characteristics demonstrated 100% sensitivity and 99% specificity at a cut-off level of 2.5 µg/mL for both SARS-CoV-2 proteins. Among 36 patients evaluated in a post-hospital follow-up clinic, median levels of IgG to spike-RBD and nucleocapsid were 34.7 µg/mL (IQR 18-52) and 24.5 µg/mL (IQR 9-59), respectively. Among 17 patients with longitudinal samples there was a wide variation in the magnitude of IgG responses, but generally the response to spike-RBD and to nucleocapsid occurred in parallel, with peak levels approaching 100 µg/mL, or 1% of total IgG. CONCLUSIONS: We have described a quantitative assay to measure IgG to SARS-CoV-2 that could be used in clinical and research laboratories and implemented at scale. The assay can easily be adapted to measure IgG to novel antigens, has good performance characteristics and a read-out in standardized units.

Unsupervised machine learning reveals key immune cell subsets in COVID-19, rhinovirus infection, and cancer therapy.

For an emerging disease like COVID-19, systems immunology tools may quickly identify and quantitatively characterize cells associated with disease progression or clinical response. With repeated sampling, immune monitoring creates a real-time portrait of the cells reacting to a novel virus before disease specific knowledge and tools are established. However, single cell analysis tools can struggle to reveal rare cells that are under 0.1% of the population. Here, the machine learning workflow Tracking Responders Expanding (T-REX) was created to identify changes in both very rare and common cells in diverse human immune monitoring settings. T-REX identified cells that were highly similar in phenotype and localized to hotspots of significant change during rhinovirus and SARS-CoV-2 infections. Specialized reagents used to detect the rhinovirus-specific CD4+ cells, MHCII tetramers, were not used during unsupervised analysis and instead 'left out' to serve as a test of whether T-REX identified biologically significant cells. In the rhinovirus challenge study, T-REX identified virus-specific CD4+ T cells based on these cells being a distinct phenotype that expanded by ≥95% following infection. T-REX successfully identified hotspots containing virus-specific T cells using pairs of samples comparing Day 7 of infection to samples taken either prior to infection (Day 0) or after clearing the infection (Day 28). Mapping pairwise comparisons in samples according to both the direction and degree of change provided a framework to compare systems level immune changes during infectious disease or therapy response. This revealed that the magnitude and direction of systemic immune change in some COVID-19 patients was comparable to that of blast crisis acute myeloid leukemia patients undergoing induction chemotherapy and characterized the identity of the immune cells that changed the most. Other COVID-19 patients instead matched an immune trajectory like that of individuals with rhinovirus infection or melanoma patients receiving checkpoint inhibitor therapy. T-REX analysis of paired blood samples provides an approach to rapidly identify and characterize mechanistically significant cells and to place emerging diseases into a systems immunology context.

Human TH1 and TH2 cells targeting rhinovirus and allergen coordinately promote allergic asthma.

BACKGROUND: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. OBJECTIVE: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. METHODS: Circulating virus-specific TH1 cells and allergen-specific TH2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. RESULTS: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1+ TH1 cells, but not allergen-specific TH2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral TH1 response in asthmatic subjects versus controls, with synchronized allergen-specific TH2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, TH2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-γ, TNF-α) correlated with both egress of circulating virus-specific TH1 cells and worse symptoms. CONCLUSIONS: Rhinovirus induces robust TH1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

Understanding the asthmatic response to an experimental rhinovirus infection: Exploring the effects of blocking IgE.

BACKGROUND: Rhinovirus frequently causes asthma exacerbations among children and young adults who are allergic. The interaction between allergen and rhinovirus-induced symptoms and inflammation over time is unclear. OBJECTIVE: Our aim was to compare the response to an experimental inoculation with rhinovirus-16 in allergic asthmatics with the response in healthy controls and to evaluate the effects of administrating omalizumab before and during the infection. METHODS: Two clinical trials were run in parallel. In one of these trials, the response to an experimental inoculation with rhinovirus-16 among asthmatics with high levels of total IgE was compared to the response in healthy controls. The other trial compared the effects of administering omalizumab versus placebo to asthmatics in a randomized, double-blind placebo-controlled investigation. The primary outcome for both trials compared lower respiratory tract symptoms (LRTSs) between study groups over the first 4 days of infection. RESULTS: Frequent comparisons of symptoms, lung function, and blood eosinophil counts revealed differences that were more pronounced among allergic asthmatics than among controls by days 2 and 3 after virus inoculation. Additionally, an augmentation of upper respiratory tract symptom scores and LRTS scores occurred among the atopic asthmatics versus the controls during the resolution of symptoms (P < .01 for upper respiratory symptom tract scores and P < .001 for LRTS scores). The beneficial effects of administering omalizumab on reducing LRTSs and improving lung function were strongest over the first 4 days. CONCLUSIONS: LRTSs and blood eosinophil counts were augmented and lung function was reduced among allergic asthmatics early after rhinovirus inoculation but increased late in the infection during symptom resolution. The effect of administering omalizumab on the response to rhinovirus was most pronounced during the early/innate phase of the infection.

T-bet+ Memory B Cells Link to Local Cross-Reactive IgG upon Human Rhinovirus Infection.

Human rhinoviruses cause the common cold and exacerbate chronic respiratory diseases. Although infection elicits neutralizing antibodies, these do not persist or cross-protect across multiple rhinovirus strains. To analyze rhinovirus-specific B cell responses in humans, we developed techniques using intact RV-A16 and RV-A39 for high-throughput high-dimensional single-cell analysis, with parallel assessment of antibody isotypes in an experimental infection model. Our approach identified T-bet+ B cells binding both viruses that account for ∼5% of CXCR5- memory B cells. These B cells infiltrate nasal tissue and expand in the blood after infection. Their rapid secretion of heterotypic immunoglobulin G (IgG) in vitro, but not IgA, matches the nasal antibody profile post-infection. By contrast, CXCR5+ memory B cells binding a single virus are clonally distinct, absent in nasal tissue, and secrete homotypic IgG and IgA, mirroring the systemic response. Temporal and spatial functions of dichotomous memory B cells might explain the ability to resolve infection while rendering the host susceptible to re-infection.

Developments in the mechanisms of allergy in 2018 through the eyes of Clinical and Experimental Allergy, Part I.

In the first of two linked articles, we describe the development in the mechanisms underlying allergy as described by Clinical & Experimental Allergy and other journals in 2018. Experimental models of allergic disease, basic mechanisms and clinical mechanisms are all covered.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

T cells in severe childhood asthma.

Severe asthma in children is a debilitating condition that accounts for a disproportionately large health and economic burden of asthma. Reasons for the lack of a response to standard anti-inflammatory therapies remain enigmatic. Work in the last decade has shed new light on the heterogeneous nature of asthma, and the varied immunopathologies of severe disease, which are leading to new treatment approaches for the individual patient. However, most studies to date that explored the immune landscape of the inflamed lower airways have focused on adults. T cells are pivotal to the inception and persistence of inflammatory processes in the diseased lungs, despite a contemporary shift in focus to immune events at the epithelial barrier. This article outlines current knowledge on the types of T cells and related cell types that are implicated in severe asthma. The potential for environmental exposures and other inflammatory cues to condition the immune environment of the lung in early life to favour pathogenic T cells and steroid resistance is discussed. The contributions of T cells and their cytokines to inflammatory processes and treatment resistance are also considered, with an emphasis on new observations in children that argue against conventional type 1 and type 2 T cell paradigms. Finally, the ability for new technologies to revolutionize our understanding of T cells in severe childhood asthma, and to guide future treatment strategies that could mitigate this disease, is highlighted.