Soluble ACE2 correlates with severe COVID-19 and can impair antibody responses.

Identifying immune modulators that impact neutralizing antibody responses against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is of great relevance. We postulated that high serum concentrations of soluble angiotensin-converting enzyme 2 (sACE2) might mask the spike and interfere with antibody maturation toward the SARS-CoV-2-receptor-binding motif (RBM). We tested 717 longitudinal samples from 295 COVID-19 patients and showed a 2- to 10-fold increase of enzymatically active sACE2 (a-sACE2), with up to 1 µg/mL total sACE2 in moderate and severe patients. Fifty percent of COVID-19 sera inhibited ACE2 activity, in contrast to 1.3% of healthy donors and 4% of non-COVID-19 pneumonia patients. A mild inverse correlation of a-sACE2 with RBM-directed serum antibodies was observed. In silico, we show that sACE2 concentrations measured in COVID-19 sera can disrupt germinal center formation and inhibit timely production of high-affinity antibodies. We suggest that sACE2 is a biomarker for COVID-19 and that soluble receptors may contribute to immune suppression informing vaccine design.

Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL + -Staphylococcus aureus infections.

Staphylococcus aureus strains that produce the toxin Panton-Valentine leukocidin (PVL-SA) frequently cause recurrent skin and soft tissue infections. PVL binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs), but the pathomechanism has not been extensively studied. Furthermore, it is unclear why some individuals colonized with PVL-SA experience recurring infections whereas others are asymptomatic. We thus aimed to (1) investigate how PVL exerts its pathogenicity on neutrophils and (2) identify factors that could help to explain the predisposition of patients with recurring infections. We provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH oxidase and reactive oxygen species production. Moreover, through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, and as such they were unable to kill S. aureus. Furthermore, we found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to be killed at a low PVL concentration than control neutrophils. Neutrophils from patients that experience recurring PVL-positive infections may thus be more sensitive to PVL-induced NET formation, which might impair their ability to combat the infection.

JAK inhibitor treatment for inborn errors of JAK/STAT signaling: An ESID/EBMT-IEWP retrospective study.

BACKGROUND: Inborn errors of immunity (IEI) with dysregulated JAK/STAT signaling present with variable manifestations of immune dysregulation and infections. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but initially reported outcomes were poor. JAK inhibitors (JAKi) offer a targeted treatment option that may be an alternative or bridge to HSCT. However, data on their current use, treatment efficacy and adverse events are limited. OBJECTIVE: We evaluated the current off-label JAKi treatment experience for JAK/STAT inborn errors of immunity (IEI) among European Society for Immunodeficiencies (ESID)/European Society for Blood and Marrow Transplantation (EBMT) Inborn Errors Working Party (IEWP) centers. METHODS: We conducted a multicenter retrospective study on patients with a genetic disorder of hyperactive JAK/STAT signaling who received JAKi treatment for at least 3 months. RESULTS: Sixty-nine patients (72% children) were evaluated (45 STAT1 gain of function [GOF], 21 STAT3-GOF, 1 STAT5B-GOF, 1 suppressor of cytokine signaling 1 [aka SOCS1] loss of function, 1 JAK1-GOF). Ruxolitinib was the predominantly prescribed JAKi (80%). Overall, treatment resulted in improvement (partial or complete remission) of clinical symptoms in 87% of STAT1-GOF and in 90% of STAT3-GOF patients. We documented highly heterogeneous dosing and monitoring regimens. The response rate and time to response varied across different diseases and manifestations. Adverse events including infection and weight gain were frequent (38% of patients) but were mild (grade I-II) and transient in most patients. At last follow-up, 52 (74%) of 69 patients were still receiving JAKi treatment, and 11 patients eventually underwent HSCT after receipt of previous JAKi bridging therapy, with 91% overall survival. CONCLUSIONS: Our study suggests that JAKi may be highly effective to treat symptomatic JAK/STAT IEI patients. Prospective studies to define optimal JAKi dosing for the variable clinical presentations and age ranges should be pursued.

Clinical and immunological characterisation of patients with common variable immunodeficiency related immune thrombocytopenia.

Primary Immune thrombocytopenia (ITP) is an autoimmune disease. Secondary ITP occurs in patients with underlying diseases such as common variable immunodeficiency (CVID). CVID is one of the most common symptomatic primary immunodeficiencies in adults, characterised by infectious and non-infectious symptoms. Amongst CVID patients, ITP is the most frequent autoimmune manifestation. In this single-centre study, we performed a clinical and immunological characterisation of 20 patients with CVID-related ITP and 20 ITP patients without CVID to compare severity and remission rates. We found that patients with CVID-related ITP had a higher WHO Bleeding Scale at initial diagnosis yet showed higher remission rates and required less treatment. Patients with ITP needed up to seven therapy options and were often treated with second-line drug therapy, whilst only one CVID-related ITP patient required second-line drug therapy. Therefore, we show that the course of thrombocytopenia in patients with CVID-related ITP is milder. Furthermore, we show that soluble interleukin-2 receptor (sIL-2R, CD25) was higher in CVID-related ITP compared to ITP patients and could accurately classify patient cohorts with an Area Under the Receiver Operating Characteristic of 0.92. Whilst none of the ITP patients had a history of immunodeficiency, we found immunological abnormalities in 12 out of 18 patients. Therefore, we recommend screening ITP patients for CVID and other immunodeficiencies to detect immune abnormalities early, as we found patients with reduced immunoglobulin levels as well as severe lymphocytopenia in our ITP cohort.

Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50.

Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-κB subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.

Skin infections due to Panton-Valentine leukocidin (PVL)-producing S. aureus-Cost effectiveness of outpatient treatment.

INTRODUCTION: Skin and soft tissue infections (SSTI) caused by Panton-Valentine leukocidin (PVL)-producing strains of Staphylococcus aureus (PVL-SA) are associated with recurrent skin abscesses. Secondary prevention, in conjunction with primary treatment of the infection, focuses on topical decolonization. Topical decolonization is a standard procedure in cases of recurrent PVL-SA skin infections and is recommended in international guidelines. However, this outpatient treatment is often not fully reimbursed by health insurance providers, which may interfere with successful PVL-SA decolonization. AIM: Our goal was to estimate the cost effectiveness of outpatient decolonization of patients with recurrent PVL-SA skin infections. We calculated the average cost of treatment for PVL-SA per outpatient decolonization procedure as well as per in-hospital stay. METHODS: The study was conducted between 2014 and 2018 at a German tertiary care university hospital. The cohort analyzed was obtained from the hospital's microbiology laboratory database. Data on medical costs, DRG-based diagnoses, and ICD-10 patient data was obtained from the hospital's financial controlling department. We calculated the average cost of treatment for patients admitted for treatment of PVL-SA induced skin infections. The cost of outpatient treatment is based on the German regulations of drug prices for prescription drugs. RESULTS: We analyzed a total of n = 466 swabs from n = 411 patients with recurrent skin infections suspected of carrying PVL-SA. PVL-SA was detected in 61.3% of all patients included in the study. Of those isolates, 80.6% were methicillin-susceptible, 19.4% methicillin-resistant. 89.8% of all patients were treated as outpatients. In 73.0% of inpatients colonized with PVL-SA the main diagnosis was SSTI. The median length of stay was 5.5 days for inpatients colonized with PVL-SA whose main diagnosis SSTI; the average cost was €2,283. The estimated costs per decolonization procedure in outpatients ranged from €50-€110, depending on the products used. CONCLUSION: Our data shows that outpatient decolonization offers a highly cost-effective secondary prevention strategy, which may prevent costly inpatient treatments. Therefore, health insurance companies should consider providing coverage of outpatient treatment of recurrent PVL-SA skin and soft tissue infections.

A Pathogenic Missense Variant in NFKB1 Causes Common Variable Immunodeficiency Due to Detrimental Protein Damage.

In common variable immunodeficiency (CVID), heterozygous damaging NFKB1 variants represent the most frequent monogenic cause. NFKB1 encodes the precursor p105, which undergoes proteasomal processing to generate the mature NF-κB transcription factor subunit p50. The majority of NFKB1 sequence changes comprises missense variants of uncertain significance (VUS), each requiring functional evaluation to assess causality, particularly in families with multiple affected members presenting with different phenotypes. In four affected members of a German family, all diagnosed with CVID, we identified a previously uncharacterized heterozygous NFKB1 missense variant (c.1049A>G; p.Tyr350Cys). The clinical phenotypes varied markedly regarding onset, frequency and severity of infections. Consistent immunologic findings were hypogammaglobulinemia with normal specific antibody response to protein- and polysaccharide-based vaccinations, reduced switched memory B cells and decreased lymphocyte proliferation upon stimulation with the B cell mitogen SAC. To assess the pathogenicity of the NFKB1 missense variant, we employed immunophenotyping and functional analyses in a routine in vitro cell culture model. Following site-directed mutagenesis to introduce the variant into overexpression vectors encoding EGFP-fused p105 or p50, we analyzed transiently transfected HEK293T cells by confocal imaging and Western blotting. The cytoplasmic p105-Tyr350Cys precursor gained only weak expression levels indicating accelerated decay. The missense change disabled processing of the precursor to prevent the generation of mutant p50. Unlike the wildtype p50, the overexpressed mutant p50-Tyr350Cys was also not sustainable and showed a conspicuous subnuclear mislocalization with accumulation in dense aggregates instead of a homogenous distribution. Electrophoretic mobility shift assays, fluorescence-based reporter gene analyses and co-transfection experiments however demonstrated, that the DNA-binding activity of p50-Tyr350Cys and the interaction with RelA(p65), IκBα and wildtype p50 were preserved. Mutation carriers had reduced p105 and p50 levels, indicating insufficient protein amounts as the most likely primary defect. In conclusion, the missense variant c.1049A>G caused a detrimental defect, preventing the persistent expression of both, the p105-Tyr350Cys precursor and the mature p50-Tyr350Cys. The variable clinical phenotypes among affected family members sharing an identical pathogenic NFKB1 variant support a disease mechanism provoked by a p105/p50 (haplo)insufficient condition.

Rituximab-Containing Treatment Regimens May Imply a Long-Term Risk for Difficult-To-Treat Chronic Hepatitis E.

Hepatitis E virus (HEV) infection is an emerging disease in industrialized countries which is usually characterized by a self-limited course. However, there is an increased risk of HEV persistence in immunocompromised risk populations, comprising patients following solid organ transplantation or hematological malignancies. Recently, chronic HEV infection following rituximab-containing treatment regimens has been described. Here we report five patients with chronic hepatitis E after prior rituximab therapy for various indications. We determined the immunological characteristics of these patients and analyzed the development of ribavirin (RBV) treatment failure-associated mutations in the HEV genome. One patient became chronically HEV-infected 110 months after administration of rituximab (RTX). Immunological characterization revealed that all patients exhibited significant hypogammaglobulinemia and CD4+ T cell lymphopenia. One patient permanently cleared HEV following weight-based ribavirin treatment while three patients failed to reach a sustained virological response. In depth mutational analysis confirmed the presence of specific mutations associated with RBV treatment failure in these patients. Our cases indicate that rituximab-containing treatment regimens might imply a relevant risk for persistent HEV infection even years after the last rituximab application. Moreover, we provide further evidence to prior observations suggesting that chronically HEV infected patients following RTX-containing treatment regimens might be difficult to treat.

Severe infections of Panton-Valentine leukocidin positive Staphylococcus aureus in children.

Infections caused by Panton-Valentine leukocidin-positive Staphylococcus aureus (PVL-SA) mostly present as recurrent skin abscesses and furunculosis. However, life-threatening infections (eg, necrotizing pneumonia, necrotizing fasciitis, and osteomyelitis) caused by PVL-SA have also been reported.We assessed the clinical phenotype, frequency, clinical implications (surgery, length of treatment in hospitals/intensive care units, and antibiotic treatments), and potential preventability of severe PVL-SA infections in children.Total, 75 children treated for PVL-SA infections in our in- and outpatient units from 2012 to 2017 were included in this retrospective study.Ten out of 75 children contracted severe infections (PVL-methicillin resistant S aureus n = 4) including necrotizing pneumonia (n = 4), necrotizing fasciitis (n = 2), pyomyositis (n = 2; including 1 patient who also had pneumonia), mastoiditis with cerebellitis (n = 1), preorbital cellulitis (n = 1), and recurrent deep furunculosis in an immunosuppressed patient (n = 1). Specific complications of PVL-SA infections were venous thrombosis (n = 2), sepsis (n = 5), respiratory failure (n = 5), and acute respiratory distress syndrome (n = 3). The median duration of hospital stay was 14 days (range 5-52 days). In 6 out of 10 patients a history suggestive for PVL-SA colonization in the patient or close family members before hospital admission was identified.PVL-SA causes severe to life-threatening infections requiring lengthy treatments in hospital in a substantial percentage of symptomatic PVL-SA colonized children. More than 50% of severe infections might be prevented by prompt testing for PVL-SA in individuals with a history of abscesses or furunculosis, followed by decolonization measures.

Altered B-cell subsets and functional B-cell defects in selective IgM deficiency.

Primary selective IgM deficiency (sIgM) is characterized by diminished serum IgM, infections and autoimmunity. Although there is some evidence of B-cell defects the pathogenesis of sIgM is poorly understood. We determined peripheral B-cell subsets and IgM-expression levels in 31 adult sIgM patients by flow cytometry. In a subset of patients B-cell subset alterations and antibody-secreting cells were determined by flow cytometry and ELISpot assay after in vitro differentiation.Patients had significantly increased transitional, decreased IgM only, switched and non-switched memory B cells and decreased membrane IgM-expression levels on memory B-cell subsets compared to healthy controls. A strongly diminished B-cell differentiation and expansion capacity was observed in 5/6 investigated patients. Severely reduced IgM-secreting capacity was detected in 2/6 patients.Taken together, our results show altered B-cell subsets and severe functional B-cell defects in sIgM. This may provide a diagnostic tool and basis for subclassification of patients to study the pathogenetic background.