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.

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein are approved for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb was purified in high yield, and it exhibited biophysical characteristics that were comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM), and it exhibited extremely high potencies against a broad array of pseudoviruses, chimeric viruses, and authentic virus variants. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

Poking COVID-19: insights on genomic constraints among immune-related genes between Qatari and Italian populations

Host genomic information, specifically genomic variations, may characterize susceptibility to disease and identify people with a higher risk of harm, leading to better targeting of care and vaccination. Italy was the epicentre for the spread of COVID-19 in Europe, the first country to go into a national lockdown and has one of the highest COVID-19 associated mortality rates. Qatar, on the other hand has a very low mortality rate. In this study, we compared whole-genome sequencing data of 14398 adults and Qatari-national to 925 Italian individuals. We also included in the comparison whole-exome sequence data from 189 Italian laboratory confirmed COVID-19 cases. We focused our study on a curated list of 3619 candidate genes involved in innate immunity and host-pathogen interaction. Two population-gene metric scores, the Delta Singleton-Cohort variant score (DSC) and Sum Singleton-Cohort variant score (SSC), were applied to estimate the presence of selective constraints in the Qatari population and in the Italian cohorts. Results based on DSC SSC metrics demonstrated a different selective pressure on three genes (MUC5AC, ABCA7, FLNA) between Qatari and Italian populations. This study highlighted the genetic differences between Qatari and Italian populations and identified a subset of genes involved in innate immunity and host-pathogen interaction.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

No evidence for allelic association between Covid-19 and ACE2 genetic variants by direct exome sequencing in 99 SARS-CoV-2 positive patients

BackgroundCoronaviruses (CoV) are a large family of viruses that are common in people and many animal species. Animal coronaviruses rarely infect humans with the exceptions of the Middle East Respiratory Syndrome (MERS-CoV), the Severe acute respiratory syndrome coronavirus (SARS-CoV), and now SARS-CoV-2, which is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19). Many studies suggested that genetic variants in ACE2 gene may influence the host susceptibility/resistance to SARS-CoV-2 virus according to the functional role of ACE2 in human pathophysiology. However, all these studies have been conducted in silico based on epidemiological and population data. We therefore investigated the occurrence of ACE2 variants in a cohort of 99 Italian unrelated individuals clinically diagnosed with coronavirus disease 19 (COVID-19) to experimental demonstrate allelic association with disease severity. MethodsBy whole-exome sequencing we analysed 99 DNA samples of severely and extremely severely COVID-19 patients hospitalized at the University Hospital of Rome "Tor Vergata" and Bambino Gesu Hospital in Rome. ResultsWe identified three different germline variants, one intronic (c.439+4G>A) and two missense (c.2158A>G, p.Asn720Asp; c.1888G>C, p.Asp630His), in 26 patients with a similar frequency between male and female and a not statistically different frequency, except for c.1888G>C, (p.Asp630His) with the ethnically matched populations (EUR). ConclusionsOur results suggest that there is not any ACE2 exonic allelic association with disease severity. It is possible that rare susceptibility alleles are located in the non-coding region of the gene able to control ACE2 gene activity. It is therefore of interest, to explore the existence of ACE2 susceptibility alleles to SARS-Co-V2 in these regulatory regions. In addition, we found no significant evidence that ACE2 alleles is associated with disease severity/sex bias in the Italian population.

Detection of an unstable non-coding tandem repeat in the ZNF291 gene.

Repeat instability is an important form of mutation that is responsible for several neurological, neurodegenerative and neuromuscular disorders. In this study we identified an unstable [CCTG](n) repeat in the second intron of the ZNF291 gene, on chromosome 15q21-24. The repeat number is polymorphic in normal population and the ZNF291 transcript is expressed in different areas of human brain, skeletal muscle and heart. These findings suggest that ZNF291 gene should be taken in consideration as an attractive candidate for neuromuscular expansion related diseases mapping in this locus.