Evaluations Of Severe Acute Respiratory Syndrome Coronavirus Therapeutics And A Viral Capacity For Plasticity And Escape.

The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emerged in 2002/2003, causing the deaths of almost a tenth of the 8000 individuals infected worldwide before it was controlled by public health measures. While the 2003 epidemic strain is likely extinct, the importance of coronaviruses as emergent zoonotic viruses was again realized with the emergence of a novel human coronavirus in Saudi Arabia in 2012. Despite a decade of research on SARS-CoV no approved vaccine or therapeutic yet exists, and development of broadly neutralizing and effective therapeutics for coronaviruses remains a priority. Neutralizing antibodies targeting the Spike glycoprotein (S) are both necessary and sufficient for protection against SARS-CoV, but the high genetic diversity and mutability of SARS-CoV in natural infections presents a challenge to both vaccine- and antibody-based therapeutics. Thus, an effective SARS-CoV therapeutic should provide S-specific immunity that is nonetheless broad enough to counter heterologous and derivative S variants. This work was designed to assess immunization strategies towards SARS-CoV, to explore the plasticity and neutralization networks of the Spike glycoprotein, and to assess the utility of molecular models to predict host range and antibody neutralization. In the first study we explored the limitations of a doubly inactivated SARS-CoV vaccine, identifying a vaccine-induced immunopathology and emphasizing the importance of rigorous challenge viruses and animal models that accurately recapitulate age-associated lung pathology. Second, in two collaborative studies we assessed multi-generational monoclonal antibodies designed to be broadly neutralizing or escape resistant, and extended our characterization of the Spike receptor binding domain (RBD) as a highly plastic antiviral target. Finally, we characterized ten recombinant Combinatorial Escape Viruses (CEVs) engineered from a database of antibody escape substitutions in the RBD. These CEVs were designed to assess the plasticity of the S-RBD, the utility of predictive modeling, and the neutralization networks across the RBD. The tools developed this study will assist in the development of predictive models and standardized platforms for combination monoclonal antibody immunotherapies for emergent viruses. These studies of SARS-CoV have extended our understanding of a key neutralizing target and have provided a valuable foundation for the rapid characterization of novel coronaviruses and potential therapeutics.Doctor of Philosoph

SARS-CoV and emergent coronaviruses: viral determinants of interspecies transmission

Most new emerging viruses are derived from strains circulating in zoonotic reservoirs. Coronaviruses, which had an established potential for cross-species transmission within domesticated animals, suddenly became relevant with the unexpected emergence of the highly pathogenic human SARS-CoV strain from zoonotic reservoirs in 2002. SARS-CoV infected approximately 8000 people worldwide before public health measures halted the epidemic. Supported by robust time-ordered sequence variation, structural biology, well-characterized patient pools, and biological data, the emergence of SARS-CoV represents one of the best studied natural models of viral disease emergence from zoonotic sources. This review article summarizes previous and more recent advances into the molecular and structural characteristics, with particular emphasis on host-receptor interactions, that drove this remarkable virus disease outbreak in human populations

Increased Antibody Affinity Confers Broad In Vitro Protection against Escape Mutants of Severe Acute Respiratory Syndrome Coronavirus

Even though the effect of antibody affinity on neutralization potency is well documented, surprisingly, its impact on neutralization breadth and escape has not been systematically determined. Here, random mutagenesis and DNA shuffling of the single-chain variable fragment of the neutralizing antibody 80R followed by bacterial display screening using anchored periplasmic expression (APEx) were used to generate a number of higher-affinity variants of the severe acute respiratory syndrome coronavirus (SARS-CoV)-neutralizing antibody 80R with equilibrium dissociation constants (KD) as low as 37 pM, a >270-fold improvement relative to that of the parental 80R single-chain variable fragment (scFv). As expected, antigen affinity was shown to correlate directly with neutralization potency toward the icUrbani strain of SARS-CoV. Additionally, the highest-affinity antibody fragment displayed 10-fold-increased broad neutralization in vitro and completely protected against several SARS-CoV strains containing substitutions associated with antibody escape. Importantly, higher affinity also led to the suppression of viral escape mutants in vitro. Escape from the highest-affinity variant required reduced selective pressure and multiple substitutions in the binding epitope. Collectively, these results support the hypothesis that engineered antibodies with picomolar dissociation constants for a neutralizing epitope can confer escape-resistant protection

A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease

Live-attenuated RNA virus vaccines are efficacious but subject to reversion to virulence. Among RNA viruses, replication fidelity is recognized as a key determinant of virulence and escape from antiviral therapy; increased fidelity is attenuating for some viruses. Coronavirus replication fidelity is approximately 20-fold greater than that of other RNA viruses and is mediated by a 3′-5′ exonuclease activity (ExoN) that likely functions in RNA proofreading. In this study, we demonstrate that engineered inactivation of SARS-CoV ExoN activity results in a stable mutator phenotype with profoundly decreased fidelity in vivo and attenuation of pathogenesis in young, aged, and immunocompromised mouse models of human SARS. The ExoN inactivation genotype and mutator phenotype are stable and do not revert to virulence, even after serial passage or long-term persistent infection in vivo. Our approach represents a strategy with potential for broad applications for the stable attenuation of coronaviruses and possibly other RNA viruses

Mechanisms of Severe Acute Respiratory Syndrome Coronavirus-Induced Acute Lung Injury

ABSTRACT Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV.IMPORTANCESevere acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, and infected patients developed an atypical pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI

Biofilm formation as a selectable phenotype for functionality of universal stress protein A in Escherichia coli

Most bacterial stress responses are highly specific to the nature of the stress. Universal Stress Protein A of Escherichia coli displays activation in response to a wide range of stresses. The protein is phosphorylated at a serine or threonine under conditions of stasis. One obstacle in the genetic analysis to elucidate a specific function of UspA has been the lack of a strong differential phenotype. We have previously investigated the ability of our strains to form biofilms in microtiter plates. In these preliminary studies, biofilm formation was shown to have promise as a selectable phenotype for uspA functionality since mutants appear to form a less robust biofilm. A Quikchange© protocol was used for site-specific mutagenesis of uspA in an attempt to determine the site(s) and necessity of phosphorylation for protein function. The biofilm assay is a modification of the OToole and Kolter (1998) assay in a 96-well polystyrene plate, staining adherent cells with crystal violet and visualizing the solubilized stain spectrophotometrically at 600nm. The uspA- deletion strain shows a significant phenotype differential from wild type (W3110) at 24 hours growth in LB, with the assay giving mutant to W3110 ratios of 0.70. In addition, paralog uspC, D, and E- deletion strains show varying strengths of biofilm formation. Four site-specific mutations of uspA have been created and confirmed by sequencing, and each shows biofilm formation comparable to a uspA+ strain. The 24 hour biofilm assay shows a reliable and significant differential between wild type E. coli and uspA mutants, making it suitable for several paths of investigation. The paralog mutants, C-, D-, and E-, have varying effects on biofilms, while four site-specific mutations of the uspA gene result in a restored biofilm formation phenotype but severely impaired viability under genotoxic stress

Biofilm formation as a selectable phenotype for functionality of universal stress protein A in Escherichia coli

Most bacterial stress responses are highly specific to the nature of the stress. Universal Stress Protein A of Escherichia coli displays activation in response to a wide range of stresses. The protein is phosphorylated at a serine or threonine under conditions of stasis. One obstacle in the genetic analysis to elucidate a specific function of UspA has been the lack of a strong differential phenotype. We have previously investigated the ability of our strains to form biofilms in microtiter plates. In these preliminary studies, biofilm formation was shown to have promise as a selectable phenotype for uspA functionality since mutants appear to form a less robust biofilm. A Quikchange© protocol was used for site--specific mutagenesis of uspA in an attempt to determine the site(s) and necessity of phosphorylation for protein function. The biofilm assay is a modification of the OToole and Kolter (1998) assay in a 96-well polystyrene plate, staining adherent cells with crystal violet and visualizing the solubilized stain spectrophotometrically at 600nm. The uspA- deletion strain shows a significant phenotype differential from wild type (W3110) at 24 hours growth in LB, with the assay giving mutant to W3110 ratios of 0.70. In addition, paralog uspC, D, and E- deletion strains show varying strengths of biofilm formation. Four site-specific mutations of uspA have been created and confirmed by sequencing, and each shows biofilm formation\ud comparable to a uspA+ strain. The 24 hour biofilm assay shows a reliable and significant differential between wild type E. coli and uspA mutants, making it suitable for several paths of investigation. The paralog mutants, C-, D-, and E-, have varying effects on biofilms, while four site-specific mutations of the uspA gene result in a restored biofilm formation phenotype but severely impaired viability under genotoxic stress