Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children.

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C.

The risk of COVID-19 death is much greater and age-dependent with type I IFN autoantibodies.

SARS-CoV-2 infection fatality rate (IFR) doubles with every five years of age from childhood onward. Circulating autoantibodies neutralizing IFN-α, IFN-ω, and/or IFN-ß are found in ~20% of deceased patients across age groups. In the general population, they are found in ~1% of individuals aged 20-70 years and in >4% of those >70 years old. With a sample of 1,261 deceased patients and 34,159 uninfected individuals, we estimated both IFR and relative risk of death (RRD) across age groups for individuals carrying autoantibodies neutralizing type I IFNs, relative to non-carriers. For autoantibodies neutralizing IFN-α2 or IFN-ω, the RRD was 17.0[95% CI:11.7-24.7] for individuals under 70 years old and 5.8[4.5-7.4] for individuals aged 70 and over, whereas, for autoantibodies neutralizing both molecules, the RRD was 188.3[44.8-774.4] and 7.2[5.0-10.3], respectively. IFRs increased with age, from 0.17%[0.12-0.31] for individuals <40 years old to 26.7%[20.3-35.2] for those ≥80 years old for autoantibodies neutralizing IFN-α2 or IFN-ω, and from 0.84%[0.31-8.28] to 40.5%[27.82-61.20] for the same two age groups, for autoantibodies neutralizing both molecules. Autoantibodies against type I IFNs increase IFRs, and are associated with high RRDs, particularly those neutralizing both IFN-α2 and -ω. Remarkably, IFR increases with age, whereas RRD decreases with age. Autoimmunity to type I IFNs appears to be second only to age among common predictors of COVID-19 death.

Replication and single-cycle delivery of SARS-CoV-2 replicons.

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.

Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths.

Circulating autoantibodies (auto-Abs) neutralizing high concentrations (10 ng/mL, in plasma diluted 1 to 10) of IFN-α and/or -ω are found in about 10% of patients with critical COVID-19 pneumonia, but not in subjects with asymptomatic infections. We detect auto-Abs neutralizing 100-fold lower, more physiological, concentrations of IFN-α and/or -ω (100 pg/mL, in 1/10 dilutions of plasma) in 13.6% of 3,595 patients with critical COVID-19, including 21% of 374 patients > 80 years, and 6.5% of 522 patients with severe COVID-19. These antibodies are also detected in 18% of the 1,124 deceased patients (aged 20 days-99 years; mean: 70 years). Moreover, another 1.3% of patients with critical COVID-19 and 0.9% of the deceased patients have auto-Abs neutralizing high concentrations of IFN-ß. We also show, in a sample of 34,159 uninfected subjects from the general population, that auto-Abs neutralizing high concentrations of IFN-α and/or -ω are present in 0.18% of individuals between 18 and 69 years, 1.1% between 70 and 79 years, and 3.4% >80 years. Moreover, the proportion of subjects carrying auto-Abs neutralizing lower concentrations is greater in a subsample of 10,778 uninfected individuals: 1% of individuals <70 years, 2.3% between 70 and 80 years, and 6.3% >80 years. By contrast, auto-Abs neutralizing IFN-ß do not become more frequent with age. Auto-Abs neutralizing type I IFNs predate SARS-CoV-2 infection and sharply increase in prevalence after the age of 70 years. They account for about 20% of both critical COVID-19 cases in the over-80s, and total fatal COVID-19 cases.

DRUL for school: Opening Pre-K with safe, simple, sensitive saliva testing for SARS-CoV-2.

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 µl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/µl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks.

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.

Auto-antibodies to type I IFNs can underlie adverse reactions to yellow fever live attenuated vaccine.

Yellow fever virus (YFV) live attenuated vaccine can, in rare cases, cause life-threatening disease, typically in patients with no previous history of severe viral illness. Autosomal recessive (AR) complete IFNAR1 deficiency was reported in one 12-yr-old patient. Here, we studied seven other previously healthy patients aged 13 to 80 yr with unexplained life-threatening YFV vaccine-associated disease. One 13-yr-old patient had AR complete IFNAR2 deficiency. Three other patients vaccinated at the ages of 47, 57, and 64 yr had high titers of circulating auto-Abs against at least 14 of the 17 individual type I IFNs. These antibodies were recently shown to underlie at least 10% of cases of life-threatening COVID-19 pneumonia. The auto-Abs were neutralizing in vitro, blocking the protective effect of IFN-α2 against YFV vaccine strains. AR IFNAR1 or IFNAR2 deficiency and neutralizing auto-Abs against type I IFNs thus accounted for more than half the cases of life-threatening YFV vaccine-associated disease studied here. Previously healthy subjects could be tested for both predispositions before anti-YFV vaccination.

Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and shared coronavirus host factors.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.

Enhanced SARS-CoV-2 neutralization by dimeric IgA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants.

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.

The new coronavirus, SARS-CoV-2, which causes the disease COVID-19, has had a serious worldwide impact on human health. The virus was virtually unknown at the beginning of 2020. Since then, intense research efforts have resulted in sequencing the coronavirus genome, identifying the structures of its proteins, and creating a wide range of tools to search for effective vaccines and therapies. Antibodies, which are immune molecules produced by the body that target specific segments of viral proteins can neutralize virus particles and trigger the immune system to kill cells infected with the virus. Several technologies are currently under development to exploit antibodies, including vaccines, blood plasma from patients who were previously infected, manufactured antibodies and more. The spike proteins on the surface of SARS-CoV-2 are considered to be prime antibody targets as they are accessible and have an essential role in allowing the virus to attach to and infect host cells. Antibodies bind to spike proteins and some can block the virus' ability to infect new cells. But some viruses, such as HIV and influenza, are able to mutate their equivalent of the spike protein to evade antibodies. It is unknown whether SARS-CoV-2 is able to efficiently evolve to evade antibodies in the same way. Weisblum, Schmidt et al. addressed this question using an artificial system that mimics natural infection in human populations. Human cells grown in the laboratory were infected with a hybrid virus created by modifying an innocuous animal virus to contain the SARS-CoV-2 spike protein, and treated with either manufactured antibodies or antibodies present in the blood of recovered COVID-19 patients. In this situation, only viruses that had mutated in a way that allowed them to escape the antibodies were able to survive. Several of the virus mutants that emerged had evolved spike proteins in which the segments targeted by the antibodies had changed, allowing these mutant viruses to remain undetected. An analysis of more than 50,000 real-life SARS-CoV-2 genomes isolated from patient samples further showed that most of these virus mutations were already circulating, albeit at very low levels in the infected human populations. These results show that SARS-CoV-2 can mutate its spike proteins to evade antibodies, and that these mutations are already present in some virus mutants circulating in the human population. This suggests that any vaccines that are deployed on a large scale should be designed to activate the strongest possible immune response against more than one target region on the spike protein. Additionally, antibody-based therapies that use two antibodies in combination should prevent the rise of viruses that are resistant to the antibodies and maintain the long-term effectiveness of vaccines and therapies.

Autoantibodies against type I IFNs in patients with life-threatening COVID-19.

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-ω (IFN-ω) (13 patients), against the 13 types of IFN-α (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men.

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.

Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses.

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.

LY6E impairs coronavirus fusion and confers immune control of viral disease.

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.

Convergent antibody responses to SARS-CoV-2 in convalescent individuals.

During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.