Environmental and genetic drivers of population differences in SARS-CoV-2 immune responses

Humans display vast clinical variability upon SARS-CoV-2 infection1-3, partly due to genetic and immunological factors4. However, the magnitude of population differences in immune responses to SARS-CoV-2 and the mechanisms underlying such variation remain unknown. Here we report single-cell RNA-sequencing data for peripheral blood mononuclear cells from 222 healthy donors of various ancestries stimulated with SARS-CoV-2 or influenza A virus. We show that SARS-CoV-2 induces a weaker, but more heterogeneous interferon-stimulated gene activity than influenza A virus, and a unique pro-inflammatory signature in myeloid cells. We observe marked population differences in transcriptional responses to viral exposure that reflect environmentally induced cellular heterogeneity, as illustrated by higher rates of cytomegalovirus infection, affecting lymphoid cells, in African-descent individuals. Expression quantitative trait loci and mediation analyses reveal a broad effect of cell proportions on population differences in immune responses, with genetic variants having a narrower but stronger effect on specific loci. Additionally, natural selection has increased immune response differentiation across populations, particularly for variants associated with SARS-CoV-2 responses in East Asians. We document the cellular and molecular mechanisms through which Neanderthal introgression has altered immune functions, such as its impact on the myeloid response in Europeans. Finally, colocalization analyses reveal an overlap between the genetic architecture of immune responses to SARS-CoV-2 and COVID-19 severity. Collectively, these findings suggest that adaptive evolution targeting immunity has also contributed to current disparities in COVID-19 risk.

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with critical COVID-19

BackgroundWe previously reported inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity in 1-5% of unvaccinated patients with life-threatening COVID-19, and auto-antibodies against type I IFN in another 15-20% of cases. MethodsWe report here a genome-wide rare variant burden association analysis in 3,269 unvaccinated patients with life-threatening COVID-19 (1,301 previously reported and 1,968 new patients), and 1,373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. A quarter of the patients tested had antibodies against type I IFN (234 of 928) and were excluded from the analysis. ResultsNo gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI:1.5-528.7, P=1.1x10-4), in analyses restricted to biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR=3.70 [95%CI:1.3-8.2], P=2.1x10-4). Adding the recently reported TYK2 COVID-19 locus strengthened this enrichment, particularly under a recessive model (OR=19.65 [95%CI:2.1-2635.4]; P=3.4x10-3). When these 14 loci and TLR7 were considered, all individuals hemizygous (n=20) or homozygous (n=5) for pLOF or bLOF variants were patients (OR=39.19 [95%CI:5.2-5037.0], P=4.7x10-7), who also showed an enrichment in heterozygous variants (OR=2.36 [95%CI:1.0-5.9], P=0.02). Finally, the patients with pLOF or bLOF variants at these 15 loci were significantly younger (mean age [SD]=43.3 [20.3] years) than the other patients (56.0 [17.3] years; P=1.68x10-5). ConclusionsRare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60 years old.

Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq

Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

Identification of driver genes for severe forms of COVID-19 in a deeply phenotyped young patient cohort

The etiopathogenesis of severe COVID-19 remains unknown. Indeed given major confounding factors (age and co-morbidities), true drivers of this condition have remained elusive. Here, we employ an unprecedented multi-omics analysis, combined with artificial intelligence, in a young patient cohort where major co-morbidities have been excluded at the onset. Here, we established a three-tier cohort of individuals younger than 50 years without major comorbidities. These included 47 "critical" (in the ICU under mechanical ventilation) and 25 "non-critical" (in a noncritical care ward) COVID-19 patients as well as 22 healthy individuals. The analyses included whole-genome sequencing, whole-blood RNA sequencing, plasma and blood mononuclear cells proteomics, cytokine profiling and high-throughput immunophenotyping. An ensemble of machine learning, deep learning, quantum annealing and structural causal modeling led to key findings. Critical patients were characterized by exacerbated inflammation, perturbed lymphoid/myeloid compartments, coagulation and viral cell biology. Within a unique gene signature that differentiated critical from noncritical patients, several driver genes promoted severe COVID-19 among which the upregulated metalloprotease ADAM9 was key. This gene signature was replicated in an independent cohort of 81 critical and 73 recovered COVID-19 patients, as were ADAM9 transcripts, soluble form and proteolytic activity. Ex vivo ADAM9 inhibition affected SARS-CoV-2 uptake and replication in human lung epithelial cells. In conclusion, within a young, otherwise healthy, COVID-19 cohort, we provide the landscape of biological perturbations in vivo where a unique gene signature differentiated critical from non-critical patients. The key driver, ADAM9, interfered with SARS-CoV-2 biology. A repositioning strategy for anti-ADAM9 therapeutic is feasible. One sentence summaryEtiopathogenesis of severe COVID19 in a young patient population devoid of comorbidities.

Antibodies against type-I Interferon: detection and association with severe clinical outcome in COVID-19 patients

ObjectivesImpairment of type I interferon (IFN-I) immunity has been reported in critically-ill COVID-19 patients. This defect can be explained in a subset of patients by the presence of circulating autoantibodies (auto-Abs) against IFN-I. We set out to improve the detection and the quantification of IFN-I auto-Abs in a cohort of critically-ill COVID-19 patients, in order to better evaluate the prevalence of these Abs as the pandemic progresses, and how they correlate with the clinical course of the disease. MethodsThe concentration of anti-IFN-2 Abs was determined in the serum of 84 critically-ill COVID-19 patients who were admitted to ICU in Hospices Civils de Lyon, France using a commercially available kit (Thermo-Fisher, Catalog #BMS217). ResultsA total of 21/84 (25%) critically-ill COVID-19 patients had circulating anti-IFN-2 Abs above cut-off (>34 ng.mL-1). Among them, 15/21 had Abs with neutralizing activity against IFN-2, i.e. 15/84 (18%) of critically-ill patients. In addition, we noticed an impairment of the IFN-I response in the majority of patients with neutralizing anti-IFN-2 Abs. There was no significant difference in the clinical characteristics or outcome of with or without neutralizing anti-IFN-2 auto-Abs. We detected anti-IFN-2 auto-Abs in COVID-19 patients sera throughout their ICU stay. Finally, we also found auto-Abs against multiple subtypes of IFN-I including IFN-{omega}. ConclusionsWe reported that 18% of critically-ill COVID-19 patients were positive for IFN-I auto-Abs, confirming that the presence of these antibodies is associated with higher risk of developing a criticall COVID-19 form.

Longitudinal single-cell epitope and RNA-sequencing reveals the immunological impact of type 1 interferon autoantibodies in critical COVID-19

Type I interferon (IFN-I) neutralizing autoantibodies have been found in some critical COVID-19 patients; however, their prevalence and longitudinal dynamics across the disease severity scale, and functional effects on circulating leukocytes remain unknown. Here, in 284 COVID-19 patients, we found IFN-I autoantibodies in 19% of critical, 6% of severe and none of the moderate cases. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 COVID-19 patients, 15 non-COVID-19 patients and 11 non-hospitalized healthy controls, revealed a lack of IFN-I stimulated gene (ISG-I) response in myeloid cells from critical cases, including those producing anti-IFN-I autoantibodies. Moreover, surface protein analysis showed an inverse correlation of the inhibitory receptor LAIR-1 with ISG-I expression response early in the disease course. This aberrant ISG-I response in critical patients with and without IFN-I autoantibodies, supports a unifying model for disease pathogenesis involving ISG-I suppression via convergent mechanisms.

A common TMPRSS2 variant protects against severe COVID-19

Infection with SARS-CoV-2 has a wide range of clinical presentations, from asymptomatic to life-threatening. Old age is the strongest factor associated with increased COVID19-related mortality, followed by sex and pre-existing conditions. The importance of genetic and immunological factors on COVID19 outcome is also starting to emerge, as demonstrated by population studies and the discovery of damaging variants in genes controlling type I IFN immunity and of autoantibodies that neutralize type I IFNs. The human protein transmembrane protease serine type 2 (TMPRSS2) plays a key role in SARS-CoV-2 infection, as it is required to activate the virus spike protein, facilitating entry into target cells. We focused on the only common TMPRSS2 non-synonymous variant predicted to be damaging (rs12329760), which has a minor allele frequency of [~]25% in the population. In a large population of SARS-CoV-2 positive patients, we show that this variant is associated with a reduced likelihood of developing severe COVID19 (OR 0.87, 95%CI:0.79-0.97, p=0.01). This association was stronger in homozygous individuals when compared to the general population (OR 0.65, 95%CI:0.50-0.84, p=1.3x10-3). We demonstrate in vitro that this variant, which causes the amino acid substitution valine to methionine, impacts the catalytic activity of TMPRSS2 and is less able to support SARS-CoV-2 spike-mediated entry into cells. TMPRSS2 rs12329760 is a common variant associated with a significantly decreased risk of severe COVID19. Further studies are needed to assess the expression of the TMPRSS2 across different age groups. Moreover, our results identify TMPRSS2 as a promising drug target, with a potential role for camostat mesilate, a drug approved for the treatment of chronic pancreatitis and postoperative reflux esophagitis, in the treatment of COVID19. Clinical trials are needed to confirm this.

A monocyte/dendritic cell molecular signature of SARS-CoV2-related multisystem inflammatory syndrome in children (MIS-C) with severe myocarditis

SARS-CoV-2 infection in children is generally milder than in adults, yet a proportion of cases result in hyperinflammatory conditions often including myocarditis. To better understand these cases, we applied a multi-parametric approach to the study of blood cells of 56 children hospitalized with suspicion of SARS-CoV-2 infection. The most severe forms of MIS-C (multisystem inflammatory syndrome in children related to SARS-CoV-2), that resulted in myocarditis, were characterized by elevated levels of pro-angiogenesis cytokines and several chemokines. Single-cell transcriptomic analyses identified a unique monocyte/dendritic cell gene signature that correlated with the occurrence of severe myocarditis, characterized by sustained NF-{kappa}B activity, TNF- signaling, associated with decreased gene expression of NF-{kappa}B inhibitors. We also found a weak response to type-I and type-II interferons, hyperinflammation and response to oxidative stress related to increased HIF-1 and VEGF signaling. These results provide potential for a better understanding of disease pathophysiology.

SARS-CoV-2 induces activation and diversification of human plasmacytoid pre-dendritic cells.

Several studies have analyzed antiviral immune pathways in late-stage severe COVID-19. However, the initial steps of SARS-CoV-2 antiviral immunity are poorly understood. Here, we have isolated primary SARS-CoV-2 viral strains, and studied their interaction with human plasmacytoid pre-dendritic cells (pDC), a key player in antiviral immunity. We show that pDC are not productively infected by SARS-CoV-2. However, they efficiently diversified into activated P1-, P2-, and P3-pDC effector subsets in response to viral stimulation. They expressed CD80, CD86, CCR7, and OX40 ligand at levels similar to influenza virus-induced activation. They rapidly produced high levels of interferon-, interferon-{lambda}1, IL-6, IP-10, and IL-8. All major aspects of SARS-CoV-2-induced pDC activation were inhibited by hydroxychloroquine. Mechanistically, SARS-CoV-2-induced pDC activation critically depended on IRAK4 and UNC93B1, as established using pDC from genetically deficient patients. Overall, our data indicate that human pDC are efficiently activated by SARS-CoV-2 particles and may thus contribute to type I IFN-dependent immunity against SARS-CoV-2 infection.

Endemic human coronaviruses induce distinct antibody repertoires in adults and children

Four endemic human coronaviruses (HCoVs) are commonly associated with acute respiratory infection in humans. B cell responses to these "common cold" viruses remain incompletely understood. Here we report a comprehensive analysis of CoV-specific antibody repertoires in 231 children and 1168 adults using phage-immunoprecipitation sequencing. Seroprevalence of antibodies to endemic HCoVs ranged between ~4 and 27% depending on the species and cohort. We identified at least 136 novel linear B cell epitopes. Antibody repertoires against endemic HCoVs were qualitatively different between children and adults in that anti-HCoV IgG specificities more frequently found among children targeted functionally important and structurally conserved regions of the spike, nucleocapsid and matrix proteins. Moreover, antibody specificities targeting the highly conserved fusion peptide region and S2 cleavage site of the spike protein were broadly cross-reactive with peptides of epidemic human and non-human coronaviruses. In contrast, an acidic tandem repeat in the N-terminal region of the Nsp3 subdomain of the HCoV-HKU1 polyprotein was the predominant target of antibody responses in adult donors. Our findings shed light on the dominant species-specific and pan-CoV target sites of human antibody responses to coronavirus infection, thereby providing important insights for the development of prophylactic or therapeutic monoclonal antibodies and vaccine design.

Outbreak of Kawasaki disease in children during COVID-19 pandemic: a prospective observational study in Paris, France

BackgroundAcute clinical manifestations of SARS-CoV-2 infection are less frequent and less severe in children than in adults. However, recent observations raised concerns about potential post-viral severe inflammatory reactions in children infected with SARS-CoV-2. MethodsWe describe an outbreak of cases of Kawasaki disease (KD) admitted between April 27 and May 7, 2020, in the general paediatrics department of a university hospital in Paris, France. All children prospectively underwent nasopharyngeal swabs for SARS-CoV-2 RT-PCR, SARS-CoV-2 IgG serology testing, and echocardiography. The number of admissions for KD during the study period was compared to that observed since January 1, 2018, based on discharge codes, using Poisson regression. ResultsA total of 17 children were admitted for KD over an 11-day period, in contrast with a mean of 1.0 case per 2-week period over 2018-2019 (Poisson incidence rate ratio: 13.2 [95% confidence interval: 7.3-24.1], p <0.001). Their median age was 7.5 (range, 3.7-16.6) years, and 59% of patients originated from sub-Saharan Africa or Caribbean islands. Eleven patients presented with KD shock syndrome (KDSS) requiring intensive care support, and 12 had myocarditis. All children had marked gastrointestinal symptoms at the early stage of illness and high levels of inflammatory markers. Fourteen patients (82%) had evidence of recent SARS-CoV-2 infection (positive RT-PCR 7/17, positive IgG antibody detection 14/16). All patients received immunoglobulins and some received corticosteroids (5/17). The clinical outcome was favourable in all patients. Moderate coronary artery dilations were detected in 5 cases (29%) during hospitalisation. ConclusionsThe ongoing outbreak of KD in the Paris might be related to SARS-CoV2, and shows an unusually high proportion of children with gastrointestinal involvement, KDSS and African ancestry.

Chronic Granulomatous Disease: An unreported mutation

@#Chronic Granulomatous Disease (CGD) is caused by defects in the phagocyte NADPH oxidase and occurs in approximately 1:200,000 births worldwide. It presents with early onset of severe recurrent bacterial and fungal infections. This is a case of a 9-year old male with severe, recurrent bacterial infections since 3 weeks of age. Initial Nitroblue tetrazolium (NBT) reduction tests were normal but a DNA analysis revealed a previously unreported homozygous mutation in CYBB, p.S418Y. Dihydrorhodamine (DHR) test showed poor neutrophil oxidation consistent with X-linked CGD. Definitive microbiologic diagnosis is essential for directing therapy for recurrent bacterial and fungal infections. Treatment of infections should be aggressive. Lifelong bacterial and fungal prophylaxis is necessary for prolonged survival. We report a case of confirmed CGD with the previously unreported mutation.

ISG15: leading a double life as a secreted molecule

ISG15 is a well-known intracellular ubiquitin-like molecule involved in ISGylation. However, a recent study has revived the notion first put forward two decades ago that ISG15 is also a secreted molecule. Human neutrophils, monocytes and lymphocytes can release ISG15, even though this protein has no detectable signal peptide sequence. ISG15 has also been found in the secretory granules of granulocytes. The mechanism underlying ISG15 secretion is unknown. Secreted ISG15 acts on at least T and natural killer (NK) lymphocytes, in which it induces interferon (IFN)-gamma production. However, the mechanism by which ISG15 stimulates these cells also remains unclear. ISG15 and IFN-gamma seem to define an innate circuit that operates preferentially, but not exclusively, between granulocytes and NK cells. Inherited ISG15 deficiency is associated with severe mycobacterial disease in both mice and humans. This infectious phenotype probably results from the lack of secreted ISG15, because patients and mice with other inborn errors of IFN-gamma immunity also display mycobacterial diseases. In addition to raising mechanistic issues, the studies described here pave the way for clinical studies of various aspects, ranging from the use of recombinant ISG15 in patients with infectious diseases to the use of ISG15-blocking agents in patients with inflammatory diseases.