RESUMO
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 (K(D)) 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.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/química , Anticorpos Antivirais/genética , Anticorpos Antivirais/farmacologia , Afinidade de Anticorpos , Linhagem Celular , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/química , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Alinhamento de Sequência , Síndrome Respiratória Aguda Grave/virologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologiaRESUMO
Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.
Assuntos
Pulmão/imunologia , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Vacinas de Produtos Inativados/uso terapêutico , Vacinas Virais/uso terapêutico , Animais , Anticorpos Antivirais/imunologia , Western Blotting , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Feminino , Pulmão/patologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Eosinofilia Pulmonar/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/prevenção & controle , Síndrome Respiratória Aguda Grave/virologia , Vacinação , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
UNLABELLED: 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. IMPORTANCE: Severe 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.
Assuntos
Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/virologia , Interações Hospedeiro-Patógeno , Pulmão/patologia , Pulmão/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Coagulação Sanguínea , Modelos Animais de Doenças , Fibrinólise , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteoma/análise , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
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 (CoV) replication fidelity is approximately 20-fold greater than that of other RNA viruses and is mediated by a 3'â5' exonuclease (ExoN) activity that probably functions in RNA proofreading. In this study we demonstrate that engineered inactivation of severe acute respiratory syndrome (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 mice. 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. ExoN inactivation has potential for broad applications in the stable attenuation of CoVs and, perhaps, other RNA viruses.
Assuntos
Exorribonucleases/antagonistas & inibidores , Exorribonucleases/metabolismo , Hospedeiro Imunocomprometido/imunologia , Síndrome Respiratória Aguda Grave/tratamento farmacológico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Vacinas Virais/farmacologia , Replicação Viral/fisiologia , Fatores Etários , Animais , Sequência de Bases , Primers do DNA/genética , Desenho de Fármacos , Exorribonucleases/genética , Exorribonucleases/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Análise de Sequência de DNA , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Estatísticas não ParamétricasRESUMO
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 hostreceptor interactions, that drove this remarkable virus disease outbreak in human populations.