Intracellular detection of virus infections is a critical component of innate immunity carried out by molecules known as pathogen recognition receptors (PRRs). Activation of PRRs by their respective ...pathogen-associated molecular patterns (PAMPs) leads to production of proinflamatory cytokines, including type I IFN, and the establishment of an antiviral state in the host. Out of all PRRs found to date, retinoic acid inducible gene (RIG-I) has been shown to play a key role in recognition of RNA viruses. On the basis of in vitro and transfection studies, 5′ppp RNA produced during virus replication is thought to bind and activate this important sensor. However, the nature of RNA molecules that interact with endogenous RIG-I during the course of viral infection has not been determined. In this work we use next-generation RNA sequencing to show that RIG-I preferentially associates with shorter, 5′ppp containing viral RNA molecules in infected cells. We found that during Sendai infection RIG-I specifically bound the genome of the defective interfering (DI) particle and did not bind the full-length virus genome or any other viral RNAs. In influenza-infected cells RIG-I preferentially associated with shorter genomic segments as well as subgenomic DI particles. Our analysis for the first time identifies RIG-I PAMPs under natural infection conditions and implies that full-length genomes of single segmented RNA virus families are not bound by RIG-I during infection.
Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically ...humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.
The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral ...form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.
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•The SARS-CoV-2 D614G S protein variant supplanted the ancestral virus in people•D614G increases infectivity on human lung cells or cells with bat or pangolin ACE2•D614G is potently neutralized by antibodies targeting the receptor-binding domain•D614G shifts S protein conformation toward an ACE2-binding fusion-competent state
Structural and molecular insights into the SARS-CoV-2 spike protein variant D614G reveal the basis of its increased infectivity
RIG-I-like receptors (RLRs: RIG-I, MDA5 and LGP2) play a major role in the innate immune response against viral infections and detect patterns on viral RNA molecules that are typically absent from ...host RNA. Upon RNA binding, RLRs trigger a complex downstream signaling cascade resulting in the expression of type I interferons and proinflammatory cytokines. In the past decade extensive efforts were made to elucidate the nature of putative RLR ligands. In vitro and transfection studies identified 5'-triphosphate containing blunt-ended double-strand RNAs as potent RIG-I inducers and these findings were confirmed by next-generation sequencing of RIG-I associated RNAs from virus-infected cells. The nature of RNA ligands of MDA5 is less clear. Several studies suggest that double-stranded RNAs are the preferred agonists for the protein. However, the exact nature of physiological MDA5 ligands from virus-infected cells needs to be elucidated. In this work, we combine a crosslinking technique with next-generation sequencing in order to shed light on MDA5-associated RNAs from human cells infected with measles virus. Our findings suggest that RIG-I and MDA5 associate with AU-rich RNA species originating from the mRNA of the measles virus L gene. Corresponding sequences are poorer activators of ATP-hydrolysis by MDA5 in vitro, suggesting that they result in more stable MDA5 filaments. These data provide a possible model of how AU-rich sequences could activate type I interferon signaling.
The cytosolic pathogen sensor RIG‐I is activated by RNAs with exposed 5′‐triphosphate (5′‐ppp) and terminal double‐stranded structures, such as those that are generated during viral infection. RIG‐I ...has been shown to translocate on dsRNA in an ATP‐dependent manner. However, the precise role of the ATPase activity in RIG‐I activation remains unclear. Using in vitro‐transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG‐I oligomerizes on 5′‐ppp dsRNA in an ATP hydrolysis‐dependent and dsRNA length‐dependent manner, which correlates with the strength of type‐I interferon (IFN‐I) activation. These results establish a clear role for the ligand‐induced ATPase activity of RIG‐I in the stimulation of the IFN response.
This study shows that RIG‐I oligomerizes on Sendai virus‐derived RNA in an ATP hydrolysis and dsRNA length dependent manner. Oligomerization correlates with the strength of interferon activation, thus identifying the role of the elusive RIG‐I ATPase activity.
Retinoic acid inducible gene I (RIG-I) is a pattern recognition receptor (PRR) responsible for detection of nucleic acids from pathogens in the cytoplasm of infected cells and induction of type I ...interferon (IFN). RIG-I -specific pathogen associated molecular patterns (PAMPs) are characterized by RNA molecules with a 5'-triphosphate (5'-ppp) group and partial double-stranded composition. Although many RNA molecules capable of activating RIG-I have been described, the exact nature of viral RNAs which are responsible for triggering RIG-I activity during the course of an infection has not been extensively explored and the specificity of RIG-I for various viral RNA molecules remains largely unknown. By examining endogenous RIG-I/RNA complexes in influenza virus and Sendai virus infected cells we were able to identify viral RNA molecules which specifically associated with RIG-I during infection. We showed that in Sendai virus infected cells, RIG-I specifically and preferentially associated with the copy-back defective interfering (DI) particle RNA and not with the full-length Sendai virus genome or Sendai virus encoded mRNAs. In influenza virus infected cells RIG-I also preferentially associated with DI RNAs as well as with the shorter genomic segments.
Retinoic acid inducible gene (RIG-I)-mediated innate immunity plays a pivotal role in defence against virus infections. Previously we have shown that Sendai virus (SeV) defective interfering (DI) RNA ...functions as an exclusive and potent RIG-I ligand in DI-RNA-rich SeV-Cantell infected cells. To further understand how RIG-I is activated during SeV infection, we used a different interferon (IFN)-inducing SeV strain, recombinant SeVΔC, which, in contrast to SeV-Cantell is believed to stimulate IFN production due to the lack of the SeV IFN antagonist protein C. Surprisingly, we found that in SevΔC-infected cells, DI RNAs also functioned as an exclusive RIG-I ligand. Infections with wild-type SeV failed to generate any RIG-I-associated immunostimulatory RNA and this correlated with the lack of DI genomes in infected cells, as well as with the absence of cellular innate immune responses. Supplementation of the C protein in the context of SeVΔC infection led to a reduction in the number of DI RNAs, further supporting the potential role of the C protein as a negative regulator of DI generation and/or accumulation. Our findings indicate that limiting DI genome production is an important function of viral IFN antagonist proteins.
REGN‐EB3 (Inmazeb) is a cocktail of three human monoclonal antibodies approved for treatment of Ebola infection. This paper describes development of a mathematical model linking REGN‐EB3’s inhibition ...of Ebola virus to survival in a non‐human primate (NHP) model, and translational scaling to predict survival in humans. Pharmacokinetic/pharmacodynamic data from single‐ and multiple‐dose REGN‐EB3 studies in infected rhesus macaques were incorporated. Using discrete indirect response models, the antiviral mechanism of action was used as a forcing function to drive the reversal of key Ebola disease hallmarks over time, for example, liver and kidney damage (elevated alanine ALT and aspartate aminotransferases AST, blood urea nitrogen BUN, and creatinine), and hemorrhage (decreased platelet count). A composite disease characteristic function was introduced to describe disease severity and integrated with the ordinary differential equations estimating the time course of clinical biomarkers. Model simulation results appropriately represented the concentration‐dependence of the magnitude and time course of Ebola infection (viral and pathophysiological), including time course of viral load, ALT and AST elevations, platelet count, creatinine, and BUN. The model estimated the observed survival rate in rhesus macaques and the dose of REGN‐EB3 required for saturation of the pharmacodynamic effects of viral inhibition, reversal of Ebola pathophysiology, and survival. The model also predicted survival in clinical trials with appropriate scaling to humans. This mathematical investigation demonstrates that drug‐disease modeling can be an important translational tool to integrate preclinical data from an NHP model recapitulating disease progression to guide future translation of preclinical data to clinical study design.
Neutralizing antibodies are key determinants of protection from future infection, yet well-validated high-throughput assays for measuring titers of SARS-CoV-2-neutralizing antibodies are not ...generally available. Here, we describe the development and validation of IMMUNO-COV v2.0, a scalable surrogate virus assay, which titrates antibodies that block infection of Vero-ACE2 cells by a luciferase-encoding vesicular stomatitis virus displaying SARS-CoV-2 spike glycoproteins (VSV-SARS2-Fluc). Antibody titers, calculated using a standard curve consisting of stepped concentrations of SARS-CoV-2 spike monoclonal antibody, correlated closely (
< 0.0001) with titers obtained from a gold standard 50% plaque-reduction neutralization test (PRNT50%) performed using a clinical isolate of SARS-CoV-2. IMMUNO-COV v2.0 was comprehensively validated using data acquired from 242 assay runs performed over 7 days by five analysts, utilizing two separate virus lots, and 176 blood samples. Assay performance was acceptable for clinical use in human serum and plasma based on parameters including linearity, dynamic range, limit of blank and limit of detection, dilutional linearity and parallelism, precision, clinical agreement, matrix equivalence, clinical specificity and sensitivity, and robustness. Sufficient VSV-SARS2-Fluc virus reagent has been banked to test 5 million clinical samples. Notably, a significant drop in IMMUNO-COV v2.0 neutralizing antibody titers was observed over a 6-month period in people recovered from SARS-CoV-2 infection. Together, our results demonstrate the feasibility and utility of IMMUNO-COV v2.0 for measuring SARS-CoV-2-neutralizing antibodies in vaccinated individuals and those recovering from natural infections. Such monitoring can be used to better understand what levels of neutralizing antibodies are required for protection from SARS-CoV-2 and what booster dosing schedules are needed to sustain vaccine-induced immunity.
Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals. A key component of the protective immune response is the production of neutralizing antibodies capable of preventing future SARS-CoV-2 infection. Yet, fundamental questions remain regarding the longevity of neutralizing antibody responses following infection or vaccination and the level of neutralizing antibodies required to confer protection. Our work is significant as it describes the development and validation of a scalable clinical assay that measures SARS-CoV-2-neutraling antibody titers. We have critical virus reagent to test over 5 million samples, making our assay well suited for widespread monitoring of SARS-CoV-2-neutralizing antibodies, which can in turn be used to inform vaccine dosing schedules and answer fundamental questions regarding SARS-CoV-2 immunity.
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