RESUMEN
The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.
Asunto(s)
COVID-19 , Humanos , Animales , Ratones , ARN Interferente Pequeño/genética , COVID-19/terapia , SARS-CoV-2/genética , Antivirales/farmacología , Antivirales/uso terapéutico , Oligonucleótidos , PulmónRESUMEN
BACKGROUND: Severe coronavirus disease 2019 (Covid-19) is associated with dysregulated inflammation. The effects of combination treatment with baricitinib, a Janus kinase inhibitor, plus remdesivir are not known. METHODS: We conducted a double-blind, randomized, placebo-controlled trial evaluating baricitinib plus remdesivir in hospitalized adults with Covid-19. All the patients received remdesivir (≤10 days) and either baricitinib (≤14 days) or placebo (control). The primary outcome was the time to recovery. The key secondary outcome was clinical status at day 15. RESULTS: A total of 1033 patients underwent randomization (with 515 assigned to combination treatment and 518 to control). Patients receiving baricitinib had a median time to recovery of 7 days (95% confidence interval [CI], 6 to 8), as compared with 8 days (95% CI, 7 to 9) with control (rate ratio for recovery, 1.16; 95% CI, 1.01 to 1.32; P = 0.03), and a 30% higher odds of improvement in clinical status at day 15 (odds ratio, 1.3; 95% CI, 1.0 to 1.6). Patients receiving high-flow oxygen or noninvasive ventilation at enrollment had a time to recovery of 10 days with combination treatment and 18 days with control (rate ratio for recovery, 1.51; 95% CI, 1.10 to 2.08). The 28-day mortality was 5.1% in the combination group and 7.8% in the control group (hazard ratio for death, 0.65; 95% CI, 0.39 to 1.09). Serious adverse events were less frequent in the combination group than in the control group (16.0% vs. 21.0%; difference, -5.0 percentage points; 95% CI, -9.8 to -0.3; P = 0.03), as were new infections (5.9% vs. 11.2%; difference, -5.3 percentage points; 95% CI, -8.7 to -1.9; P = 0.003). CONCLUSIONS: Baricitinib plus remdesivir was superior to remdesivir alone in reducing recovery time and accelerating improvement in clinical status among patients with Covid-19, notably among those receiving high-flow oxygen or noninvasive ventilation. The combination was associated with fewer serious adverse events. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT04401579.).
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/uso terapéutico , Azetidinas/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Purinas/uso terapéutico , Pirazoles/uso terapéutico , Sulfonamidas/uso terapéutico , Adenosina Monofosfato/efectos adversos , Adenosina Monofosfato/uso terapéutico , Adulto , Anciano , Alanina/efectos adversos , Alanina/uso terapéutico , Antivirales/efectos adversos , Azetidinas/efectos adversos , COVID-19/mortalidad , COVID-19/terapia , Método Doble Ciego , Quimioterapia Combinada , Femenino , Mortalidad Hospitalaria , Hospitalización , Humanos , Inhibidores de las Cinasas Janus/efectos adversos , Inhibidores de las Cinasas Janus/uso terapéutico , Masculino , Persona de Mediana Edad , Terapia por Inhalación de Oxígeno , Purinas/efectos adversos , Pirazoles/efectos adversos , Respiración Artificial , Sulfonamidas/efectos adversos , Resultado del TratamientoRESUMEN
BACKGROUND: Although several therapeutic agents have been evaluated for the treatment of coronavirus disease 2019 (Covid-19), no antiviral agents have yet been shown to be efficacious. METHODS: We conducted a double-blind, randomized, placebo-controlled trial of intravenous remdesivir in adults who were hospitalized with Covid-19 and had evidence of lower respiratory tract infection. Patients were randomly assigned to receive either remdesivir (200 mg loading dose on day 1, followed by 100 mg daily for up to 9 additional days) or placebo for up to 10 days. The primary outcome was the time to recovery, defined by either discharge from the hospital or hospitalization for infection-control purposes only. RESULTS: A total of 1062 patients underwent randomization (with 541 assigned to remdesivir and 521 to placebo). Those who received remdesivir had a median recovery time of 10 days (95% confidence interval [CI], 9 to 11), as compared with 15 days (95% CI, 13 to 18) among those who received placebo (rate ratio for recovery, 1.29; 95% CI, 1.12 to 1.49; P<0.001, by a log-rank test). In an analysis that used a proportional-odds model with an eight-category ordinal scale, the patients who received remdesivir were found to be more likely than those who received placebo to have clinical improvement at day 15 (odds ratio, 1.5; 95% CI, 1.2 to 1.9, after adjustment for actual disease severity). The Kaplan-Meier estimates of mortality were 6.7% with remdesivir and 11.9% with placebo by day 15 and 11.4% with remdesivir and 15.2% with placebo by day 29 (hazard ratio, 0.73; 95% CI, 0.52 to 1.03). Serious adverse events were reported in 131 of the 532 patients who received remdesivir (24.6%) and in 163 of the 516 patients who received placebo (31.6%). CONCLUSIONS: Our data show that remdesivir was superior to placebo in shortening the time to recovery in adults who were hospitalized with Covid-19 and had evidence of lower respiratory tract infection. (Funded by the National Institute of Allergy and Infectious Diseases and others; ACTT-1 ClinicalTrials.gov number, NCT04280705.).
Asunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/uso terapéutico , Infecciones por Coronavirus/tratamiento farmacológico , Neumonía Viral/tratamiento farmacológico , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/efectos adversos , Adenosina Monofosfato/uso terapéutico , Administración Intravenosa , Adulto , Anciano , Alanina/administración & dosificación , Alanina/efectos adversos , Alanina/uso terapéutico , Antivirales/administración & dosificación , Antivirales/efectos adversos , Betacoronavirus , COVID-19 , Infecciones por Coronavirus/mortalidad , Infecciones por Coronavirus/terapia , Método Doble Ciego , Oxigenación por Membrana Extracorpórea , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Terapia por Inhalación de Oxígeno , Pandemias , Neumonía Viral/mortalidad , Neumonía Viral/terapia , Respiración Artificial , SARS-CoV-2 , Factores de Tiempo , Adulto Joven , Tratamiento Farmacológico de COVID-19RESUMEN
Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.
Asunto(s)
Farmacorresistencia Viral , Subtipo H1N1 del Virus de la Influenza A , Oseltamivir , Proteínas Virales , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia Viral/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H1N1 del Virus de la Influenza A/genética , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , Virus de la Influenza A/genética , Gripe Humana/tratamiento farmacológico , Mutación , Neuraminidasa/genética , Neuraminidasa/metabolismo , Oseltamivir/farmacología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
[Figure: see text].
Asunto(s)
Enzima Convertidora de Angiotensina 2/metabolismo , Apoptosis , Plaquetas/metabolismo , COVID-19/metabolismo , Serina Endopeptidasas/metabolismo , Células A549 , Adulto , Plaquetas/ultraestructura , Plaquetas/virología , Western Blotting , COVID-19/sangre , COVID-19/virología , Femenino , Interacciones Huésped-Patógeno , Humanos , Masculino , Microscopía Electrónica de Transmisión , Necroptosis , SARS-CoV-2/fisiologíaRESUMEN
RIG-I and MDA5 have emerged as key cytosolic sensors for the detection of RNA viruses and lead to antiviral interferon (IFN) production. Recent studies have highlighted the importance of posttranslational modifications for controlling RIG-I antiviral activity. However, the regulation of MDA5 signal-transducing ability remains unclear. Here, we show that MDA5 signaling activity is regulated by a dynamic balance between phosphorylation and dephosphorylation of its caspase recruitment domains (CARDs). Employing a phosphatome RNAi screen, we identified PP1α and PP1γ as the primary phosphatases that are responsible for MDA5 and RIG-I dephosphorylation and that lead to their activation. Silencing of PP1α and PP1γ enhanced RIG-I and MDA5 CARD phosphorylation and reduced antiviral IFN-ß production. PP1α- and PP1γ-depleted cells were impaired in their ability to induce IFN-stimulated gene expression, which resulted in enhanced RNA virus replication. This work identifies PP1α and PP1γ as regulators of antiviral innate immune responses to various RNA viruses, including influenza virus, paramyxovirus, dengue virus, and picornavirus.
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ARN Helicasas DEAD-box/inmunología , Inmunidad Innata/inmunología , Proteína Fosfatasa 1/inmunología , ARN Viral/inmunología , Animales , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunidad Innata/genética , Immunoblotting , Helicasa Inducida por Interferón IFIH1 , Interferón beta/inmunología , Interferón beta/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Interferencia de ARN , ARN Viral/genética , ARN Viral/metabolismo , Receptores Inmunológicos , Transducción de Señal/genética , Transducción de Señal/inmunología , Células VeroRESUMEN
Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.
Asunto(s)
Farmacorresistencia Viral , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Mutación , Neuraminidasa/genética , Animales , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Vacunas contra la Influenza , Células de Riñón Canino Madin Darby , Modelos Moleculares , Neuraminidasa/química , Pruebas de Neutralización , Genética Inversa , Análisis de Secuencia de ARN , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Pimodivir, a first-in-class inhibitor of influenza virus polymerase basic protein 2, is being developed for hospitalized and high-risk patients with influenza A. METHODS: In this double-blinded phase 2b study, adults with acute uncomplicated influenza A were randomized 1:1:1:1 to receive one of the following treatments twice daily for 5 days: placebo, pimodivir 300 mg or 600 mg, or pimodivir 600 mg plus oseltamivir 75 mg. Antiviral activity, safety, and pharmacokinetics of pimodivir alone or in combination were evaluated. RESULTS: Of 292 patients randomized, 223 were treated and had confirmed influenza A virus infection. The trial was stopped early because the primary end point was met; the area under the curve of the viral load, determined by quantitative reverse transcription-polymerase chain reaction analysis, in nasal secretions from baseline to day 8 significantly decreased in the active treatment groups, compared with the placebo group (300 mg group, -3.6 day*log10 copies/mL [95% confidence interval {CI}, -7.1 to -0.1]; 600 mg group, -4.5 [95%CI -8.0 to -1.0]; and combination group, -8.6 [95% CI, -12.0 to -5.1]). Pimodivir plus oseltamivir yielded a significantly lower viral load titer over time than placebo and a trend for a shorter time to symptom resolution than placebo. Pimodivir plasma concentrations increased in a dose-proportional manner. The most commonly reported adverse event was mild or moderate diarrhea. CONCLUSIONS: Pimodivir (with or without oseltamivir) resulted in significant virologic improvements over placebo, demonstrated trends in clinical improvement, and was well tolerated. Pimodivir 600 mg twice daily is in further development. CLINICAL TRIALS REGISTRATION: NCT02342249, 2014-004068-39, and CR107745.
Asunto(s)
Antivirales/uso terapéutico , Virus de la Influenza A , Gripe Humana/tratamiento farmacológico , Oseltamivir/uso terapéutico , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Pirroles/uso terapéutico , Enfermedad Aguda , Adulto , Antivirales/efectos adversos , Antivirales/sangre , Antivirales/farmacocinética , Diarrea/inducido químicamente , Método Doble Ciego , Farmacorresistencia Viral/genética , Quimioterapia Combinada , Terminación Anticipada de los Ensayos Clínicos , Femenino , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Gripe Humana/virología , Masculino , Persona de Mediana Edad , Oseltamivir/sangre , Piridinas/efectos adversos , Piridinas/sangre , Piridinas/farmacocinética , Pirimidinas/efectos adversos , Pirimidinas/sangre , Pirimidinas/farmacocinética , Pirroles/efectos adversos , Pirroles/sangre , Pirroles/farmacocinética , Factores de Tiempo , Carga Viral , Proteínas Virales/genética , Esparcimiento de VirusRESUMEN
In this issue of Immunity, Town et al. (2009) show the interplay between Toll-like receptors (TLRs) and cytokines during West Nile virus infection and define a role for TLR-mediated production of interleukin-23 in immune cell homing and pathogenesis.
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Receptores Toll-Like/inmunología , Virus del Nilo Occidental/inmunología , Animales , Interleucina-23/biosíntesis , Interleucina-23/inmunología , ARN Helicasas/metabolismoRESUMEN
Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.
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Subtipo H1N1 del Virus de la Influenza A/genética , Caperuzas de ARN/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Regulación Viral de la Expresión Génica , Genes Virales , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.
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Farmacorresistencia Viral/genética , Genética de Población , Subtipo H1N1 del Virus de la Influenza A/genética , Selección Genética , Teorema de Bayes , Flujo Genético , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/genética , Gripe Humana/virología , Mutación , Oseltamivir/farmacologíaRESUMEN
We report a case of probable Zaire Ebola virus-related ophthalmologic complications in a physician from the United States who contracted Ebola virus disease in Liberia. Uveitis, immune activation, and nonspecific increase in antibody titers developed during convalescence. This case highlights immune phenomena that could complicate management of Ebola virus disease-related uveitis during convalescence.
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Ebolavirus , Fiebre Hemorrágica Ebola/complicaciones , Fiebre Hemorrágica Ebola/virología , Uveítis/diagnóstico , Uveítis/etiología , Ebolavirus/genética , Fiebre Hemorrágica Ebola/diagnóstico , Humanos , Liberia , Masculino , Tomografía de Coherencia Óptica , Uveítis/tratamiento farmacológicoRESUMEN
Influenza A virus (IAV) has a segmented genome that allows for the exchange of genome segments between different strains. This reassortment accelerates evolution by breaking linkage, helping IAV cross species barriers to potentially create highly virulent strains. Challenges associated with monitoring the process of reassortment in molecular detail have limited our understanding of its evolutionary implications. We applied a novel deep sequencing approach with quantitative analysis to assess the in vitro temporal evolution of genomic reassortment in IAV. The combination of H1N1 and H3N2 strains reproducibly generated a new H1N2 strain with the hemagglutinin and nucleoprotein segments originating from H1N1 and the remaining six segments from H3N2. By deep sequencing the entire viral genome, we monitored the evolution of reassortment, quantifying the relative abundance of all IAV genome segments from the two parent strains over time and measuring the selection coefficients of the reassorting segments. Additionally, we observed several mutations coemerging with reassortment that were not found during passaging of pure parental IAV strains. Our results demonstrate how reassortment of the segmented genome can accelerate viral evolution in IAV, potentially enabled by the emergence of a small number of individual mutations.
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Alphainfluenzavirus/genética , Genoma Viral , Virus Reordenados/genética , Selección Genética , Animales , Biología Computacional , Perros , Evolución Molecular , Frecuencia de los Genes , Genotipo , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Límite de Detección , Células de Riñón Canino Madin Darby , Nucleoproteínas/genética , Análisis de Secuencia de ARNRESUMEN
Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.
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Antivirales/farmacología , Farmacorresistencia Viral/genética , Evolución Molecular , Genoma Viral , Subtipo H1N1 del Virus de la Influenza A/genética , Oseltamivir/farmacología , Animales , Línea Celular , Perros , Ensayos Analíticos de Alto Rendimiento , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Concentración 50 Inhibidora , Mutación , Ensayo de Placa ViralRESUMEN
Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, causes acute infectious mononucleosis (AIM) and is linked to the development of several human malignancies. There is an urgent need for a vaccine that is safe, prevents infection and/or limits disease. Unique among human herpesviruses, glycoprotein (gp)350/220, which initiates EBV attachment to susceptible host cells, is the major ligand on the EBV envelope and is highly conserved. Interaction between gp350/220 and complement receptor type 2 (CR2)/CD21 and/or (CR1)/CD35 on B-cells is required for infection. Potent antibody responses to gp350/220 occur in animal models and humans. Thus, gp350/220 provides an attractive candidate for prophylactic subunit vaccine development. However, in a recent Phase II clinical trial immunization with soluble recombinant gp350 reduced the incidence of AIM, but did not prevent infection. Despite various attempts to produce an EBV vaccine, no vaccine is licensed. Herein we describe a sub-unit vaccine against EBV based on a novel Newcastle disease virus (NDV)-virus-like particle (VLP) platform consisting of EBVgp350/220 ectodomain fused to NDV-fusion (F) protein. The chimeric protein EBVgp350/220-F is incorporated into the membrane of a VLP composed of the NDV matrix and nucleoprotein. The particles resemble native EBV in diameter and shape and bind CD21 and CD35. Immunization of BALB/c mice with EBVgp350/220-F VLPs elicited strong, long-lasting neutralizing antibody responses when assessed in vitro. This chimeric VLP is predicted to provide a superior safety profile as it is efficiently produced in Chinese hamster ovary (CHO) cells using a platform devoid of human nucleic acid and EBV-transforming genes.
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Anticuerpos Neutralizantes/biosíntesis , Linfocitos B/citología , Proteínas Recombinantes/metabolismo , Proteínas de la Matriz Viral/inmunología , Virión/metabolismo , Animales , Antígenos CD/metabolismo , Adhesión Celular , Línea Celular , Humanos , Inmunización , Inmunoglobulina G/metabolismo , Ratones Endogámicos BALB C , Pruebas de Neutralización , Unión ProteicaRESUMEN
Polymeric microparticles have been widely investigated as platforms for delivery of drugs, vaccines, and imaging contrast agents and are increasingly used in a variety of clinical applications. Microparticles activate the inflammasome complex and induce the processing and secretion of IL-1ß, a key innate immune cytokine. Recent work suggests that although receptors are clearly important for particle phagocytosis, other physical characteristics, especially shape, play an important role in the way microparticles activate cells. We examined the role of particle surface texturing not only on uptake efficiency but also on the subsequent immune cell activation of the inflammasome. Using a method based on emulsion processing of amphiphilic block copolymers, we prepared microparticles with similar overall sizes and surface chemistries but having either smooth or highly microtextured surfaces. In vivo, textured (budding) particles induced more rapid neutrophil recruitment to the injection site. In vitro, budding particles were more readily phagocytosed than smooth particles and induced more lipid raft recruitment to the phagosome. Remarkably, budding particles also induced stronger IL-1ß secretion than smooth particles through activation of the NLRP3 inflammasome. These findings demonstrate a pronounced role of particle surface topography in immune cell activation, suggesting that shape is a major determinant of inflammasome activation.
Asunto(s)
Inmunidad Innata , Inflamasomas/inmunología , Polímeros , Animales , Proteínas Portadoras/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Neutrófilos/inmunología , Neutrófilos/metabolismo , Tamaño de la Partícula , Fagocitosis/inmunología , Polímeros/química , Transducción de Señal , Propiedades de SuperficieRESUMEN
Influenza is a severe disease in humans and animals with few effective therapies available. All strains of influenza virus are prone to developing drug resistance due to the high mutation rate in the viral genome. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of influenza. Influenza uses many individually weak ligand binding interactions for a high avidity multivalent attachment to sialic acid-bearing cells. Polymerized sialic acid analogs can form multivalent interactions with influenza but are not ideal therapeutics due to solubility and toxicity issues. We used liposomes as a novel means for delivery of the glycan sialylneolacto-N-tetraose c (LSTc). LSTc-bearing decoy liposomes form multivalent, polymer-like interactions with influenza virus. Decoy liposomes competitively bind influenza virus in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. Inhibition is specific for influenza virus, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind influenza virus or inhibit infectivity. LSTc decoy liposomes prevent the spread of influenza virus during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. LSTc decoy liposomes co-localize with fluorescently tagged influenza virus, whereas control liposomes do not. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high avidity interactions with influenza hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging influenza strains.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Polisacáridos/farmacología , Ácidos Siálicos/farmacología , Animales , Antivirales/administración & dosificación , Línea Celular , Chlorocebus aethiops , Perros , Evaluación Preclínica de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/virología , Femenino , Hemaglutinación/efectos de los fármacos , Humanos , Virus de la Influenza A/fisiología , Liposomas , Ratones , Ratones Endogámicos C57BL , Polisacáridos/administración & dosificación , Virus del Sarcoma de Rous/efectos de los fármacos , Virus Sendai/efectos de los fármacos , Ácidos Siálicos/administración & dosificación , Células Vero , Replicación Viral/efectos de los fármacosRESUMEN
Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412-423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5-1.0 log(10) reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/uso terapéutico , Hepatitis C Crónica/terapia , Hepatitis C/prevención & control , Secuencia de Aminoácidos , Animales , Línea Celular , Modelos Animales de Enfermedad , Hepatitis C/inmunología , Hepatitis C/virología , Hepatitis C Crónica/inmunología , Humanos , Trasplante de Hígado , Mutación , Pruebas de Neutralización , Pan troglodytes , ARN Viral/sangre , Tetraspanina 28/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Carga ViralRESUMEN
Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.
Asunto(s)
Proteínas ADAM , Infecciones por Cardiovirus , Virus de la Encefalomiocarditis , Inmunidad Innata , Interferón Tipo I , Helicasa Inducida por Interferón IFIH1 , Proteínas de la Membrana , Miocarditis , Animales , Ratones , Proteínas ADAM/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Infecciones por Cardiovirus/inmunología , Infecciones por Cardiovirus/virología , Virus de la Encefalomiocarditis/inmunología , Células HEK293 , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/inmunología , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones Endogámicos C57BL , Ratones Noqueados , Miocarditis/inmunología , Miocarditis/virología , Transducción de Señal/inmunologíaRESUMEN
Prepatellar bursitis is typically a monomicrobial bacterial infection. A fungal cause is rarely identified. We describe a 61-year-old man who had received a renal transplant 21 months prior to presentation whose synovial fluid and surgical specimens grew Phomopsis bougainvilleicola, a pycnidial coelomycete.