Identification of new drugs to counteract anti-spike IgG-induced hyperinflammation in severe COVID-19

Previously, we and others have shown that SARS-CoV-2 spike-specific IgG antibodies play a major role in disease severity in COVID-19 by triggering macrophage hyperactivation, disrupting endothelial barrier integrity, and inducing thrombus formation. This hyperinflammation is dependent on high levels of anti-spike IgG with aberrant Fc tail glycosylation, leading to Fcγ receptor hyperactivation. For development of immune-regulatory therapeutics, drug specificity is crucial to counteract excessive inflammation whereas simultaneously minimizing the inhibition of antiviral immunity. We here developed an in vitro activation assay to screen for small molecule drugs that specifically counteract antibody-induced pathology. We identified that anti-spike-induced inflammation is specifically blocked by small molecule inhibitors against SYK and PI3K. We identified SYK inhibitor entospletinib as the most promising candidate drug, which also counteracted anti-spike-induced endothelial dysfunction and thrombus formation. Moreover, entospletinib blocked inflammation by different SARS-CoV-2 variants of concern. Combined, these data identify entospletinib as a promising treatment for severe COVID-19

The effect of immunosuppressive therapies on the endothelial host response in critically ill COVID-19 patients

While several effective therapies for critically ill patients with COVID-19 have been identified in large, well-conducted trials, the mechanisms underlying these therapies have not been investigated in depth. Our aim is to investigate the association between various immunosuppressive therapies (corticosteroids, tocilizumab and anakinra) and the change in endothelial host response over time in critically ill COVID-19 patients. We conducted a pre-specified multicenter post-hoc analysis in a Dutch cohort of COVID-19 patients admitted to the ICU between March 2020 and September 2021 due to hypoxemic respiratory failure. A panel of 18 immune response biomarkers in the complement, coagulation and endothelial function domains were measured using ELISA or Luminex. Biomarkers were measured on day 0-1, day 2-4 and day 6-8 after start of COVID-19 treatment. Patients were categorized into four treatment groups: no immunomodulatory treatment, corticosteroids, anakinra plus corticosteroids, or tocilizumab plus corticosteroids. The association between treatment group and the change in concentrations of biomarkers was estimated with linear mixed-effects models, using no immunomodulatory treatment as reference group. 109 patients with a median age of 62 years [IQR 54-70] of whom 72% (n = 78) was male, were included in this analysis. Both anakinra plus corticosteroids (n = 22) and tocilizumab plus corticosteroids (n = 38) were associated with an increase in angiopoietin-1 compared to no immune modulator (n = 23) (beta of 0.033 [0.002-0.064] and 0.041 [0.013-0.070] per day, respectively). These treatments, as well as corticosteroids alone (n = 26), were further associated with a decrease in the ratio of angiopoietin-2/angiopoietin-1 (beta of 0.071 [0.034-0.107], 0.060 [0.030-0.091] and 0.043 [0.001-0.085] per day, respectively). Anakinra plus corticosteroids and tocilizumab plus corticosteroids were associated with a decrease in concentrations of complement complex 5b-9 compared to no immunomodulatory treatment (0.038 [0.006-0.071] and 0.023 [0.000-0.047], respectively). Currently established treatments for critically ill COVID-19 patients are associated with a change in biomarkers of the angiopoietin and complement pathways, possibly indicating a role for stability of the endothelium. These results increase the understanding of the mechanisms of interventions and are possibly useful for stratification of patients with other inflammatory conditions which may potentially benefit from these treatments

Targeted exhaled breath analysis for detection of Pseudomonas aeruginosa in cystic fibrosis patients

Background: Pseudomonas aeruginosa (PA) is an important respiratory pathogen for cystic fibrosis (CF) patients. Routine microbiology surveillance is time-consuming, and is best performed on expectorated sputum. As alternative, volatile organic compounds (VOCs) may be indicative of PA colonisation. In this study, we aimed to identify VOCs associated with PA in literature and perform targeted exhaled breath analysis to recognize PA positive CF patients non-invasively. Methods: This study consisted of 1) a literature review to select VOCs of interest, and 2) a cross-sectional CF study. Definitions used: A) PA positive, PA culture at visit/chronically; B) PA free, no PA culture in ≥12 months. Exhaled VOCs were identified via quadrupole MS. The primary endpoint was the area under the receiver operating characteristics curve (AUROCC) of individual VOCs as well as combined VOCs against PA culture. Results: 241 VOCs were identified in literature, of which 56 were further evaluated, and 13 could be detected in exhaled breath in our cohort. Exhaled breath of 25 pediatric and 28 adult CF patients, PA positive (n=16) and free (n=28) was available. 3/13 VOCs were significantly (p<0.05) different between PA groups in children; none were in adults. Notably, a composite model based on 5 or 1 VOC(s) showed an AUROCC of 0.86 (CI 0.71–1.0) and 0.87 (CI 0.72–1.0) for adults and children, respectively. Conclusions: Targeted VOC analysis appears to discriminate children and adults with and without PA positive cultures with clinically acceptable sensitivity values

Targeted exhaled breath analysis for detection of Pseudomonas aeruginosa in cystic fibrosis patients

Background: Pseudomonas aeruginosa (PA) is an important respiratory pathogen for cystic fibrosis (CF) patients. Routine microbiology surveillance is time-consuming, and is best performed on expectorated sputum. As alternative, volatile organic compounds (VOCs) may be indicative of PA colonisation. In this study, we aimed to identify VOCs associated with PA in literature and perform targeted exhaled breath analysis to recognize PA positive CF patients non-invasively. Methods: This study consisted of 1) a literature review to select VOCs of interest, and 2) a cross-sectional CF study. Definitions used: A) PA positive, PA culture at visit/chronically; B) PA free, no PA culture in ≥12 months. Exhaled VOCs were identified via quadrupole MS. The primary endpoint was the area under the receiver operating characteristics curve (AUROCC) of individual VOCs as well as combined VOCs against PA culture. Results: 241 VOCs were identified in literature, of which 56 were further evaluated, and 13 could be detected in exhaled breath in our cohort. Exhaled breath of 25 pediatric and 28 adult CF patients, PA positive (n=16) and free (n=28) was available. 3/13 VOCs were significantly (p<0.05) different between PA groups in children; none were in adults. Notably, a composite model based on 5 or 1 VOC(s) showed an AUROCC of 0.86 (CI 0.71–1.0) and 0.87 (CI 0.72–1.0) for adults and children, respectively. Conclusions: Targeted VOC analysis appears to discriminate children and adults with and without PA positive cultures with clinically acceptable sensitivity values

eNose breath prints as a surrogate biomarker for classifying patients with asthma by atopy

Background: Electronic noses (eNoses) are emerging point-of-care tools that may help in the subphenotyping of chronic respiratory diseases such as asthma. Objective: We aimed to investigate whether eNoses can classify atopy in pediatric and adult patients with asthma. Methods: Participants with asthma and/or wheezing from 4 independent cohorts were included; BreathCloud participants (n = 429), Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes adults (n = 96), Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes pediatric participants (n = 100), and Pharmacogenetics of Asthma Medication in Children: Medication with Anti-Inflammatory Effects 2 participants (n = 30). Atopy was defined as a positive skin prick test result (≥3 mm) and/or a positive specific IgE level (≥0.35 kU/L) for common allergens. Exhaled breath profiles were measured by using either an integrated eNose platform or the SpiroNose. Data were divided into 2 training and 2 validation sets according to the technology used. Supervised data analysis involved the use of 3 different machine learning algorithms to classify patients with atopic versus nonatopic asthma with reporting of areas under the receiver operating characteristic curves as a measure of model performance. In addition, an unsupervised approach was performed by using a bayesian network to reveal data-driven relationships between eNose volatile organic compound profiles and asthma characteristics. Results: Breath profiles of 655 participants (n = 601 adults and school-aged children with asthma and 54 preschool children with wheezing [68.2% of whom were atopic]) were included in this study. Machine learning models utilizing volatile organic compound profiles discriminated between atopic and nonatopic participants with areas under the receiver operating characteristic curves of at least 0.84 and 0.72 in the training and validation sets, respectively. The unsupervised approach revealed that breath profiles classifying atopy are not confounded by other patient characteristics. Conclusion: eNoses accurately detect atopy in individuals with asthma and wheezing in cohorts with different age groups and could be used in asthma phenotyping

Targeted exhaled breath analysis for detection of Pseudomonas aeruginosa in cystic fibrosis patients

Background Pseudomonas aeruginosa (PA) is an important respiratory pathogen for cystic fibrosis (CF) patients. Routine microbiology surveillance is time-consuming, and is best performed on expectorated sputum. As alternative, volatile organic compounds (VOCs) may be indicative of PA colonisation. In this study, we aimed to identify VOCs associated with PA in literature and perform targeted exhaled breath analysis to recognize PA positive CF patients non-invasively. Methods This study consisted of 1) a literature review to select VOCs of interest, and 2) a cross-sectional CF study. Definitions used: A) PA positive, PA culture at visit/chronically; B) PA free, no PA culture in ≥12 months. Exhaled VOCs were identified via quadrupole MS. The primary endpoint was the area under the receiver operating characteristics curve (AUROCC) of individual VOCs as well as combined VOCs against PA culture. Results 241 VOCs were identified in literature, of which 56 were further evaluated, and 13 could be detected in exhaled breath in our cohort. Exhaled breath of 25 pediatric and 28 adult CF patients, PA positive (n=16) and free (n=28) was available. 3/13 VOCs were significantly (p<0.05) different between PA groups in children; none were in adults. Notably, a composite model based on 5 or 1 VOC(s) showed an AUROCC of 0.86 (CI 0.71–1.0) and 0.87 (CI 0.72–1.0) for adults and children, respectively. Conclusions Targeted VOC analysis appears to discriminate children and adults with and without PA positive cultures with clinically acceptable sensitivity values

Inborn errors of type I IFN immunity in patients with life-threatening COVID-19

Clinical outcome upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ranges from silent infection to lethal coronavirus disease 2019 (COVID-19). We have found an enrichment in rare variants predicted to be loss-of-function (LOF) at the 13 human loci known to govern Toll-like receptor 3 (TLR3)- and interferon regulatory factor 7 (IRF7)-dependent type I interferon (IFN) immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5%) 17 to 77 years of age. We show that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3-and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection

Lack of association between classical HLA genes and asymptomatic SARS-CoV-2 infection

Human genetic studies of critical COVID-19 pneumonia have revealed the essential role of type I interferon-dependent innate immunity to SARS-CoV-2 infection. Conversely, an association between the HLA-B∗15:01 allele and asymptomatic SARS-CoV-2 infection in unvaccinated individuals was recently reported, suggesting a contribution of pre-existing T cell-dependent adaptive immunity. We report a lack of association of classical HLA alleles, including HLA-B∗15:01, with pre-omicron asymptomatic SARS-CoV-2 infection in unvaccinated participants in a prospective population-based study in the United States (191 asymptomatic vs. 945 symptomatic COVID-19 cases). Moreover, we found no such association in the international COVID Human Genetic Effort cohort (206 asymptomatic vs. 574 mild or moderate COVID-19 cases and 1,625 severe or critical COVID-19 cases). Finally, in the Human Challenge Characterisation study, the three HLA-B∗15:01 individuals infected with SARS-CoV-2 developed symptoms. As with other acute primary infections studied, no classical HLA alleles favoring an asymptomatic course of SARS-CoV-2 infection were identified

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5–10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2–46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0–7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (Fc gamma RIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger Fc gamma RIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.Proteomic