SIGLEC1 enables straightforward assessment of type I interferon activity in inflammatory myopathies

ObjectiveTo evaluate SIGLEC1 expression on monocytes by flow cytometry as a type I interferon biomarker in idiopathic inflammatory myopathies (IIM).MethodsWe performed a retrospective analysis of adult and pediatric patients with the diagnosis of IIM. SIGLEC1 expression was assessed by flow cytometry and was compared with Physician Global Assessment (PGA) or Childhood Myositis Assessment Scale (CMAS) disease activity scores. Mann Whitney-U test and receiver operating characteristic (ROC) curves were used for cross-sectional data analysis (n=96), two-level mixed-effects linear regression model for longitudinal analyses (n=26, 110 visits). Response to treatment was analyzed in 14 patients within 12 months, using Wilcoxon test. SIGLEC1 was compared to ISG15/MxA status by immunohistochemical staining of muscle biopsies (n=17).Results96 patients with adult (a) and juvenile (j) dermatomyositis (DM, n=38), antisynthetase syndrome (AS, n=19), immune-mediated necrotizing myopathy (IMNM, n=8), inclusion body myositis (IBM, n=9), and overlap myositis (n=22) were included. SIGLEC1 distinguished significantly between active and inactive disease with an area under the curve (AUC) of 0.92 (95% CI: 0.83–1) in DM and correlated with disease activity longitudinally (aDM: standardized beta=0.54, p<0.001; jDM: standardized beta=-0.70, p<0.001). Response to treatment in DM was associated with a decreasing SIGLEC1 (p<0.01, Wilcoxon test). A positive ISG15/MxA stain was highly associated with a SIGLEC1 upregulation. SIGLEC1 was found upregulated in AS (42.1%) and IBM (22,2%) and not in IMNM.ConclusionSIGLEC1 is a candidate biomarker to assess type I interferon activity in IIM and proved useful for monitoring disease activity and response to treatment in juvenile and adult DM.Key messagesWhat is already known about this subject?There is an unmet need for routine clinical biomarkers to assess type I interferon activity in rheumatic musculoskeletal diseasesSIGLEC1 is part of the type I interferon signature and transcripts were found to be upregulated in various autoimmune diseases such as systemic lupus erythematosus (SLE), Sjoegren syndrome and dermatomyositis.SIGLEC1 is expressed on the surface of monocytes and thus is easily assessable by flow cytometry, which enables straightforward monitoring of type I interferon activityWhat does this study add?An activation of the type I interferon system in IIM can be identified by flow cytometry analysing SIGLEC1 expression. SIGLEC1 correlated to disease activity and improvement under therapy in juvenile and adult dermatomyositis.How might this impact on clinical practice or future developments?SIGLEC1 expression is a suitable biomarker for monitoring type I interferon activation in rheumatic musculoskeletal diseases, which has clinical implications for patient stratification and treatment decision making especially in the context of interferon inhibitory therapies.

476 AK119, a humanized anti-CD73 monoclonal antibody, as Immunotherapy for COVID-19

BackgroundCD73, an ecto-5’-nucleotidase involved in ATP metabolism, converts AMP into adenosine. ATP could induce production of interferon-beta1, which induces cellular resistance to viral infection and triggers apoptosis of virus-infected cells.2 In COVID-19, SARS-CoV-2 causes severe respiratory syndrome by effectively inhibiting interferon activity,3 4 leading to impaired anti-viral response. Moreover, lung injury seen in severe COVID-19 patients might be caused by excess adenosine.5 Inhibition of CD73 is believed to increase extracellular ATP, thereby countering COVID-19. Furthermore, independent of its inhibitory effects on CD73, preclinical results from other anti-CD73 mAb suggested CD73 blockade activates lymphocytes, induced antibody production from B cells and enhanced lymphocyte trafficking,6 thereby, stimulated the production of anti-SARS-CoV-2 antibodies leading to the rapid clearance of the virus.7 We developed AK119, a humanized monoclonal antibody targeting CD73, as an immunotherapy agent for the treatment of COVID-19.MethodsEvaluation of AK119 activity to bind to the CD73 antigen was performed by using ELISA, Fortebio, and FACS assay. The activity of AK119 to inhibit enzymatic activity of CD73 was evaluated by cell-based enzyme assay; and the activity of AK119 to induce internalization of CD73 and enhance CD69 and CD83 expression on B cell were performed by using FACS assays. We also investigated the potential of AK119 to promote immunoglobulin production from human B cells.ResultsAK119 could effectively bind to human CD73 with high affinity, which is comparable or superior to 10.3AA, a leading anti-CD73 antibody, in protein-based assays. AK119 inhibited CD73 enzymatic activity on MDA-MB-231 cells (IC50_AK119, 27.60 nM; IC50_10.3AA, 15.99 nM) and U87-MG cells (IC50_AK119, 0.2448 nM; IC50_10.3AA, 0.0691 nM), with a higher maximal inhibition rates of 108.26% in MDA-MB-231 cells and 96.24% in U87-MG cells compared with 10.3AA (77.02% and 75.77%, respectively). AK119 effectively induced CD73 internalization in MDA-MB-231 cells and U87-MG cells, and the internalization rate of CD73 was 60.75% and 82.39%, respectively; for 10.3AA, the internalization rate was 50.53% and 73.65%, respectively. Moreover, AK119 could stimulate approximately 4–5 fold up-regulation of CD69 (figure 1A) and CD83 (figure 1B) that are markers of B cell activation; and, AK119 significantly promoted IgG production from B cells.ConclusionsIn summary, in comparison to a leading CD73 antibody currently in clinical trial, AK119 demonstrated more complete CD73 inhibition; and more dramatic B cell activation and antibody production. Thus, A119 presented desirable preclinical bioactivities. The safety and pharmacokinetics of single ascending doses of AK119 will be evaluated in healthy volunteers in an upcoming Phase 1, First-in-Human study (NCT04516564).Trial RegistrationNCT04516564ReferencesZhang C, He H, Wang L, et al. Virus-Triggered ATP release limits viral replication through facilitating IFN-β production in a P2X7-dependent manner. J Immunol 2017;199:1372–1381.Schneider WM, Chevillotte MD, Rice CM. Interferon-stimulated genes: a complex web of host defenses. Annu Rev Immunol 2014;32:513–545.Hadjadj J, Yatim N, Barnabei L, et al. Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science 2020;369:718–724.Yuen CK, Lam JY, Wong WM, et al. SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists. Emerg Microbes Infect 2020;9:1418–1428.Hoogendijk AJ, Pinhanços SS, van der Poll T, Wieland CW. AMP-activated protein kinase activation by 5-aminoimidazole-4-carbox-amide-1-β-D-ribofuranoside (AICAR) reduces lipoteichoic acid-induced lung inflammation. J Biol Chem 2013;288:7047–7052.Luke, Jason J., et al. Immunobiology, preliminary safety, and efficacy of CPI-006, an anti-CD73 antibody with immune modulating activity, in a phase 1 trial in advanced cancers. J Clin Oncol 2019;37: suppl 2505–2505.Corvus Pharmaceuticals, Inc. Corvus Pharmaceuticals Provides Business Update and Reports Second Quarter 2020 Financial Results. 30 July 2020. [https://corvuspharma.gcs-web.com/news-releases/news-release-details/corvus-pharmaceuticals-provides-business-update-and-reports-4].Abstract 476 Figure 1AK119 induces up-regulation of CD69 and CD83 expression on B cells. PBMCs were incubated overnight with AK119 or 10.3AA. Flow cytometry analysis was performed with gating on B cells (CD19+CD3-) and mean fluorescence intensity (MFI) is reported for antibody staining of (A) CD69 or (B) CD83. One-way ANOVA, **P<0.01, ***P<0.001, vs Human IgG1 group.

Central nervous system immune response in postinfectious hydrocephalus

Inflammation following neonatal infection is a dominant cause of childhood hydrocephalus in the developing world. Understanding this complex inflammatory response is critical for the development of preventive therapies. In 100 African hydrocephalic infants ≤3 months of age, with and without a history of infection, we elucidated the biological pathways that account for this inflammatory response. We integrated proteomics and RNA sequencing in cerebrospinal fluid, identifying gene pathways involving neutrophil, interleukin (4, 12, and 13) and interferon activity associated with this condition. These findings are required to develop strategies to reduce the risk of hydrocephalus during treatment of infection.

Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients

Coronavirus disease 2019 (COVID-19) is characterized by distinct patterns of disease progression that suggest diverse host immune responses. We performed an integrated immune analysis on a cohort of 50 COVID-19 patients with various disease severity. A distinct phenotype was observed in severe and critical patients, consisting of a highly impaired interferon (IFN) type I response (characterized by no IFN-β and low IFN-α production and activity), which was associated with a persistent blood viral load and an exacerbated inflammatory response. Inflammation was partially driven by the transcriptional factor nuclear factor–κB and characterized by increased tumor necrosis factor–α and interleukin-6 production and signaling. These data suggest that type I IFN deficiency in the blood could be a hallmark of severe COVID-19 and provide a rationale for combined therapeutic approaches.

Characterization of Type-I IFN subtype autoantibodies and activity in SLE serum and urine

Background/objective Type-I interferons contribute to pathogenesis in systemic lupus erythematosus, including nephritis. Interferons consist of a family of 16 proteins yet are often characterized in patients without knowledge of the specific interferon subtypes involved. Different interferons may function in the kidneys, and other organs, relative to what is often measured in patient blood. Moreover, antibodies to interferons may potentially modulate systemic or organ-specific interferon activity. The aim of this study was to characterize global interferon activity levels and identify autoantibodies to the 12 interferon α subtypes in patient serum and urine. Methods Interferon activity levels in serum and urine were measured using an interferon bioassay. Anti-interferon and anti-cytokine autoantibodies were measured by ELISA. Serum and urine samples were also characterized for their ability to neutralize the biological activity of exogenously added interferons. Results Serum interferon activity was increased in 62% of systemic lupus erythematosus patient samples, relative to healthy donor controls, whereas binding interferon α autoantibodies to at least one interferon α subtype were found in 68% of the samples evaluated. High Systemic Lupus Erythematosus Disease Activity Index scores were significantly ( p = 0.001) associated with patient samples containing interferon α autoantibodies to three or more interferon α subtypes in their serum. Interferon α autoantibodies that potently block interferon activity were rare (∼5% of samples), but collectively bound to all 12 interferon α subtypes. Urine interferon activity and interferon α autoantibody profiles did not correlate with their serum counterparts, suggesting immune responses in systemic lupus erythematosus kidneys can be distinct from those measured in serum. Analysis of autoantibodies to 15 additional cytokines in serum identified higher frequencies of granulocyte-macrophage colony-stimulating factor and interleukin 17A autoantibodies, suggesting these signaling pathways may potentially contribute, with interferons, to systemic lupus erythematosus pathogenesis. Conclusions The measurement of autoantibodies to multiple interferon subtypes in serum and urine may provide an alternative method for following interferon-mediated systemic lupus erythematosus disease activity. The results suggest autoantibodies might be used for patient monitoring and/or identifying additional cytokine signaling pathways that are functioning in different systemic lupus erythematosus patients.

SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant

One of the features distinguishing SARS-CoV-2 from its more pathogenic counterpart SARS-CoV is the presence of premature stop codons in its ORF3b gene. Here, we show that SARS-CoV-2 ORF3b is a potent interferon antagonist, suppressing the induction of type I interferon more efficiently than its SARS-CoV ortholog. Phylogenetic analyses and functional assays revealed that SARS-CoV-2-related viruses from bats and pangolins also encode truncated ORF3b gene products with strong anti-interferon activity. Furthermore, analyses of more than 15,000 SARS-CoV-2 sequences identified a natural variant, in which a longer ORF3b reading frame was reconstituted. This variant was isolated from two patients with severe disease and further increased the ability of ORF3b to suppress interferon induction. Thus, our findings not only help to explain the poor interferon response in COVID-19 patients, but also describe a possibility of the emergence of natural SARS-CoV-2 quasispecies with extended ORF3b that may exacerbate COVID-19 symptoms.HighlightsORF3b of SARS-CoV-2 and related bat and pangolin viruses is a potent IFN antagonistSARS-CoV-2 ORF3b suppresses IFN induction more efficiently than SARS-CoV orthologThe anti-IFN activity of ORF3b depends on the length of its C-terminusAn ORF3b with increased IFN antagonism was isolated from two severe COVID-19 cases

Impaired type I interferon activity and exacerbated inflammatory responses in severe Covid-19 patients

BackgroundCoronavirus disease 2019 (Covid-19) is a major global threat that has already caused more than 100,000 deaths worldwide. It is characterized by distinct patterns of disease progression implying a diverse host immune response. However, the immunological features and molecular mechanisms involved in Covid-19 severity remain so far poorly known.MethodsWe performed an integrated immune analysis that included in-depth phenotypical profiling of immune cells, whole-blood transcriptomic and cytokine quantification on a cohort of fifty Covid19 patients with a spectrum of disease severity. All patient were tested 8 to 12 days following first symptoms and in absence of anti-inflammatory therapy.ResultsA unique phenotype in severe and critically ill patients was identified. It consists in a profoundly impaired interferon (IFN) type I response characterized by a low interferon production and activity, with consequent downregulation of interferon-stimulated genes. This was associated with a persistent blood virus load and an exacerbated inflammatory response that was partially driven by the transcriptional factor NFĸB. It was also characterized by increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production and signaling as well as increased innate immune chemokines.ConclusionWe propose that type-I IFN deficiency in the blood is a hallmark of severe Covid-19 and could identify and define a high-risk population. Our study provides a rationale for testing IFN administration combined with adapted anti-inflammatory therapy targeting IL-6 or TNF-α in most severe patients. These data also raise concern for utilization of drugs that interfere with the IFN pathway.