RESUMEN
The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-ß) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo.
Asunto(s)
Inmunidad Innata , Interferón beta , Macrófagos Peritoneales , Vesiculovirus , Animales , Ratones , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/virología , Macrófagos Peritoneales/metabolismo , Interferón beta/inmunología , Interferón beta/metabolismo , Interferón beta/genética , Vesiculovirus/inmunología , Vesiculovirus/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Receptores de Reconocimiento de Patrones/inmunologíaRESUMEN
Histone deacetylates family proteins have been studied for their function in regulating viral replication by deacetylating non-histone proteins. RIG-I (Retinoic acid-inducible gene I) is a critical protein in RNA virus-induced innate antiviral signaling pathways. Our previous research showed that HDAC8 (histone deacetylase 8) involved in innate antiviral immune response, but the underlying mechanism during virus infection is still unclear. In this study, we showed that HDAC8 was involved in the regulation of vesicular stomatitis virus (VSV) replication. Over-expression of HDAC8 inhibited while knockdown promoted VSV replication. Further exploration demonstrated that HDAC8 interacted with and deacetylated RIG-I, which eventually lead to enhance innate antiviral immune response. Collectively, our data clearly demonstrated that HDAC8 inhibited VSV replication by promoting RIG-I mediated interferon production and downstream signaling pathway.
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Proteína 58 DEAD Box , Histona Desacetilasas , Inmunidad Innata , Receptores Inmunológicos , Transducción de Señal , Vesiculovirus , Replicación Viral , Proteína 58 DEAD Box/metabolismo , Proteína 58 DEAD Box/genética , Humanos , Histona Desacetilasas/metabolismo , Vesiculovirus/inmunología , Receptores Inmunológicos/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Acetilación , Células HEK293 , Interferones/metabolismo , Interferones/inmunología , Línea Celular , Interacciones Huésped-Patógeno/inmunología , Animales , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
The interferon (IFN) system protects mammals from diseases caused by virus infections. IFN synthesis is induced by pattern recognition receptor signaling pathways activated by virus infection. IFN is secreted from the infected cells and acts upon neighboring cells by binding cell surface receptors and triggering induction of hundreds of IFN-stimulated genes and proteins, many of which block different steps of virus replication. The IFN-induced tetratricopeptide repeat proteins (IFIT) are a family of RNA-binding proteins. We and others have previously reported that IFIT2 protects mice from many neurotropic RNA viruses; indeed, Ifit2-/- mice are very susceptible to intranasal or subcutaneous infections with vesicular stomatitis virus (VSV). Here, using a newly generated conditional knockout mouse, we report that ablation of Ifit2 expression only in neuronal cells was sufficient to render mice susceptible to neuropathogenesis caused by intranasal, but not subcutaneous, infection of VSV. Another genetically modified mouse line, expressing a mutant IFIT2 that cannot bind RNA, was as susceptible to VSV infection as Ifit2-/- mice. These results demonstrated that IFIT2 RNA-binding activity is essential for protecting mice against neurological diseases caused by intranasal infection of VSV.IMPORTANCEInterferon's (IFN's) antiviral effects are mediated by the proteins encoded by the interferon-stimulated genes. IFN-stimulated genes (IFIT2) is one such protein, which inhibits replication of many RNA viruses in the mouse brain and the resultant neuropathology. Our study sheds light on how IFIT2 works. By ablating Ifit2 expression only in neuronal cells, using a newly generated conditional knockout mouse line, we showed that Ifit2 induction in the neurons of the infected mouse was necessary for antiviral function of interferon. IFIT2 has no known enzyme activity; instead, it functions by binding to cellular or viral proteins or RNAs. We engineered a new mouse line that expressed a mutant IFIT2 that cannot bind RNA. These mice were very susceptible to infection with vesicular stomatitis virus indicating that the RNA-binding property of IFIT2 was essential for its antiviral function in vivo.
Asunto(s)
Ratones Noqueados , Neuronas , Proteínas de Unión al ARN , Estomatitis Vesicular , Animales , Ratones , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Neuronas/virología , Neuronas/metabolismo , Estomatitis Vesicular/virología , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/genética , Replicación Viral , Vesiculovirus/inmunología , Vesiculovirus/genética , Ratones Endogámicos C57BL , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/genética , Proteínas Reguladoras de la ApoptosisRESUMEN
Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD-nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.IMPORTANCEHerpes B virus (BV) is a highly pathogenic zoonotic virus against humans. BV belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). By contrast to HSV, cell entry mechanisms of BV are not fully understood. The research procedures to manipulate infectious BV should be conducted in biosafety level (BSL)-4 facilities. As pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus, we tried to generate a pseudotyped vesicular stomatitis virus bearing BV glycoproteins (VSV/BVpv) by modification of expression constructs of BV glycoproteins, and successfully obtained VSV/BVpv with a high titer. This study has provided novel information for constructing VSV/BVpv and its usefulness to study BV infection.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Internalización del Virus , Animales , Anticuerpos Neutralizantes/inmunología , Chlorocebus aethiops , Células Vero , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Humanos , Pruebas de Neutralización , Vesiculovirus/genética , Vesiculovirus/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismo , Glicoproteínas/genética , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: Metastatic breast cancer is a leading cause of cancer death in woman. Current treatment options are often associated with adverse side effects and poor outcomes, demonstrating the need for effective new treatments. Immunotherapies can provide durable outcomes in many cancers; however, limited success has been achieved in metastatic triple negative breast cancer. We tested whether combining different immunotherapies can target metastatic triple negative breast cancer in pre-clinical models. METHODS: Using primary and metastatic 4T1 triple negative mammary carcinoma models, we examined the therapeutic effects of oncolytic vesicular stomatitis virus (VSVΔM51) engineered to express reovirus-derived fusion associated small transmembrane proteins p14 (VSV-p14) or p15 (VSV-p15). These viruses were delivered alone or in combination with natural killer T (NKT) cell activation therapy mediated by adoptive transfer of α-galactosylceramide-loaded dendritic cells. RESULTS: Treatment of primary 4T1 tumors with VSV-p14 or VSV-p15 alone increased immunogenic tumor cell death, attenuated tumor growth, and enhanced immune cell infiltration and activation compared to control oncolytic virus (VSV-GFP) treatments and untreated mice. When combined with NKT cell activation therapy, oncolytic VSV-p14 and VSV-p15 reduced metastatic lung burden to undetectable levels in all mice and generated immune memory as evidenced by enhanced in vitro recall responses (tumor killing and cytokine production) and impaired tumor growth upon rechallenge. CONCLUSION: Combining NKT cell immunotherapy with enhanced oncolytic virotherapy increased anti-tumor immune targeting of lung metastasis and presents a promising treatment strategy for metastatic breast cancer.
Asunto(s)
Células T Asesinas Naturales , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Femenino , Ratones , Células T Asesinas Naturales/inmunología , Viroterapia Oncolítica/métodos , Humanos , Línea Celular Tumoral , Virus Oncolíticos/genética , Virus Oncolíticos/inmunología , Inmunoterapia/métodos , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Neoplasias de la Mama Triple Negativas/terapia , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/patología , Terapia Combinada , Metástasis de la Neoplasia , Vesiculovirus/genética , Células Dendríticas/inmunología , Neoplasias de la Mama/terapia , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Modelos Animales de EnfermedadRESUMEN
Neonatal Fc receptor (FcRn) recycles immunoglobulin G, and inhibition of FcRn is used clinically for treatment of autoimmune diseases. In this work, using the vesicular stomatitis virus (VSV) mouse infection model system, we determined the role of FcRn during virus infection. While induction of neutralizing antibodies and long-term protection of these antibodies was hardly affected in FcRn deficient mice, FcRn deficiency limited the amount of natural IgG (VSV-specific) antibodies. Lack of natural antibodies (nAbs) limited early control of VSV in macrophages, accelerated propagation of virus in several organs, led to the spread of VSV to the neural tissue resulting in fatal outcomes. Adoptive transfer of natural IgG into FcRn deficient mice limited early propagation of VSV in FcRn deficient mice and enhanced survival of FcRn knockout mice. In line with this, vaccination of FcRn mice with very low dose of VSV prior to infection similarly prevented death after infection. In conclusion we determined the importance of nAbs during VSV infection. Lack of FcRn limited nAbs and thereby enhanced the susceptibility to virus infection.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Antígenos de Histocompatibilidad Clase I , Inmunoglobulina G , Ratones Noqueados , Receptores Fc , Estomatitis Vesicular , Animales , Ratones , Inmunoglobulina G/inmunología , Receptores Fc/inmunología , Receptores Fc/genética , Receptores Fc/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Estomatitis Vesicular/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/inmunología , Vesiculovirus/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Modelos Animales de Enfermedad , Traslado Adoptivo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BLRESUMEN
We evaluated the in vitro effects of lyophilization for 2 vesicular stomatitis virus-based vaccines by using 3 stabilizing formulations and demonstrated protective immunity of lyophilized/reconstituted vaccine in guinea pigs. Lyophilization increased stability of the vaccines, but specific vesicular stomatitis virus-based vaccines will each require extensive analysis to optimize stabilizing formulations.
Asunto(s)
Modelos Animales de Enfermedad , Liofilización , Estomatitis Vesicular , Vacunas Virales , Animales , Cobayas , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/prevención & control , Estomatitis Vesicular/virología , Vesiculovirus/inmunología , Vesiculovirus/genética , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Eficacia de las Vacunas , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
Interferon regulatory factor 3 (IRF3) is an essential transductor for initiation of many immune responses. Here, we show that lncRNA-ISIR directly binds IRF3 to promote its phosphorylation, dimerization, and nuclear translocation, along with enhanced target gene productions. In vivo lncRNA-ISIR deficiency results in reduced IFN production, uncontrolled viral replication, and increased mortality. The human homolog, AK131315, also binds IRF3 and promotes its activation. More important, AK131315 expression is positively correlated with type I interferon (IFN-I) level and severity in patients with lupus. Mechanistically, in resting cells, IRF3 is bound to suppressor protein Flightless-1 (Fli-1), which keeps its inactive state. Upon infection, IFN-I-induced lncRNA-ISIR binds IRF3 at DNA-binding domain in cytoplasm and removes Fli-1's association from IRF3, consequently facilitating IRF3 activation. Our results demonstrate that IFN-I-inducible lncRNA-ISIR feedback strengthens IRF3 activation by removing suppressive Fli-1 in immune responses, revealing a method of lncRNA-mediated modulation of transcription factor (TF) activation.
Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Macrófagos Peritoneales/metabolismo , ARN Largo no Codificante/metabolismo , Estomatitis Vesicular/metabolismo , Animales , Estudios de Casos y Controles , Chlorocebus aethiops , Modelos Animales de Enfermedad , Silenciador del Gen , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/genética , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Células RAW 264.7 , ARN Largo no Codificante/genética , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Células Vero , Estomatitis Vesicular/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/patogenicidadRESUMEN
Chemokine production by epithelial cells is important for neutrophil recruitment during viral infection, the appropriate regulation of which is critical for restraining inflammation and attenuating subsequent tissue damage. Epithelial cell expression of long noncoding RNAs (lncRNAs), RNA-binding proteins, and their functional interactions during viral infection and inflammation remain to be fully understood. Here, we identified an inducible lncRNA in the Cxcl2 gene locus, lnc-Cxcl2, which could selectively inhibit Cxcl2 expression in mouse lung epithelial cells but not in macrophages. lnc-Cxcl2-deficient mice exhibited increased Cxcl2 expression, enhanced neutrophils recruitment, and more severe inflammation in the lung after influenza virus infection. Mechanistically, nucleus-localized lnc-Cxcl2 bound to Cxcl2 promoter, recruited a ribonucleoprotein La, which inhibited the chromatin accessibility of chemokine promoters, and consequently inhibited Cxcl2 transcription in cis However, unlike mouse lnc-Cxcl2, human lnc-CXCL2-4-1 inhibited multiple immune cytokine expressions including chemokines in human lung epithelial cells. Together, our results demonstrate a self-protecting mechanism within epithelial cells to restrain chemokine and neutrophil-mediated inflammation, providing clues for better understanding chemokine regulation and epithelial cell function in lung viral infection.
Asunto(s)
Quimiocina CXCL2/genética , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , ARN Largo no Codificante/genética , Células A549 , Animales , Línea Celular Tumoral , Quimiocina CXCL2/metabolismo , Cromatina/metabolismo , Células Epiteliales/metabolismo , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Humanos , Inflamación/prevención & control , Mediadores de Inflamación , Virus de la Influenza A/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Neumonía/inmunología , Neumonía/patología , Neumonía/virología , Regiones Promotoras Genéticas/genética , Células RAW 264.7 , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/patología , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in late 2019 and has since caused a global pandemic resulting in millions of cases and deaths. Diagnostic tools and serological assays are critical for controlling the outbreak, especially assays designed to quantitate neutralizing antibody levels, considered the best correlate of protection. As vaccines become increasingly available, it is important to identify reliable methods for measuring neutralizing antibody responses that correlate with authentic virus neutralization but can be performed outside biosafety level 3 (BSL3) laboratories. While many neutralizing assays using pseudotyped virus have been developed, there have been few studies comparing the different assays to each other as surrogates for authentic virus neutralization. Here, we characterized three enzyme-linked immunosorbent assays (ELISAs) and three pseudotyped vesicular stomatitis virus (VSV) neutralization assays and assessed their concordance with authentic virus neutralization. The most accurate assays for predicting authentic virus neutralization were luciferase- and secreted embryonic alkaline phosphatase (SEAP)-expressing pseudotyped virus neutralizations, followed by green fluorescent protein (GFP)-expressing pseudotyped virus neutralization, and then the ELISAs. IMPORTANCE The ongoing COVID-19 pandemic is caused by infection with severe acute respiratory syndrome virus 2 (SARS-CoV-2). Prior infection or vaccination can be detected by the presence of antibodies in the blood. Antibodies in the blood are also considered to be protective against future infections from the same virus. The "gold standard" assay for detecting protective antibodies against SARS-CoV-2 is neutralization of authentic SARS-CoV-2 virus. However, this assay can only be performed under highly restrictive biocontainment conditions. We therefore characterized six antibody-detecting assays for their correlation with authentic virus neutralization. The significance of our research is in outlining the advantages and disadvantages of the different assays and identifying the optimal surrogate assay for authentic virus neutralization. This will allow for more accurate assessments of protective immunity against SARS-CoV-2 following infection and vaccination.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Prueba Serológica para COVID-19/métodos , COVID-19/diagnóstico , Pruebas de Neutralización/métodos , SARS-CoV-2/inmunología , Adulto , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dominios Proteicos/inmunología , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular New Jersey/inmunologíaRESUMEN
Functional tumor-specific cytotoxic T cells elicited by therapeutic cancer vaccination in combination with oncolytic viruses offer opportunities to address resistance to checkpoint blockade therapy. Two cancer vaccines, the self-adjuvanting protein vaccine KISIMA, and the recombinant oncolytic vesicular stomatitis virus pseudotyped with LCMV-GP expressing tumor-associated antigens, termed VSV-GP-TAA, both show promise as a single agent. Here we find that, when given in a heterologous prime-boost regimen with an optimized schedule and route of administration, combining KISIMA and VSV-GP-TAA vaccinations induces better cancer immunity than individually. Using several mouse tumor models with varying degrees of susceptibility for viral replication, we find that priming with KISIMA-TAA followed by VSV-GP-TAA boost causes profound changes in the tumor microenvironment, and induces a large pool of poly-functional and persistent antigen-specific cytotoxic T cells in the periphery. Combining this heterologous vaccination with checkpoint blockade further improves therapeutic efficacy with long-term survival in the spectrum. Overall, heterologous vaccination with KISIMA and VSV-GP-TAA could sensitize non-inflamed tumors to checkpoint blockade therapy.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Virus Oncolíticos/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , Antígenos de Neoplasias/administración & dosificación , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/administración & dosificación , Terapia Combinada , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral , Vacunación , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/fisiología , Replicación ViralRESUMEN
As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2'3' cyclic phosphate instead of the common 5' triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5'triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response.
Asunto(s)
Proteína 58 DEAD Box/genética , Interacciones Huésped-Patógeno/genética , Inmunidad Innata , Virus de la Influenza A/inmunología , Receptores Inmunológicos/genética , Células A549 , Proteína 58 DEAD Box/inmunología , Genoma Viral , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Virus de la Influenza A/patogenicidad , Gripe Humana/virología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Receptores Inmunológicos/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Replicación Viral/inmunologíaRESUMEN
Innate immunity is the first line of host defense against viral infection. As one of the innate immune cell types, antigen-presenting cells play an important role in the process of antiviral immunity. This protocol describes the analysis of innate immunity induced by vesicular stomatitis virus infection of peritoneal macrophages in vitro and in vivo detection of IFN-ß production and lung injury. For complete details on the use and execution of this protocol, please refer to Shen et al. (2021).
Asunto(s)
Separación Celular/métodos , Inmunidad Innata/fisiología , Virosis/diagnóstico por imagen , Animales , Células Presentadoras de Antígenos/inmunología , Interferón Tipo I/inmunología , Macrófagos/inmunología , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Virosis/inmunología , Replicación Viral/inmunologíaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Vacunas contra la COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adulto , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/genética , Femenino , Células HEK293 , Humanos , Sueros Inmunes/inmunología , Masculino , Persona de Mediana Edad , Mutación , Pruebas de Neutralización , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Vacunación/métodos , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/inmunología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.
Asunto(s)
Anticuerpos Neutralizantes/análisis , Anticuerpos Antivirales/análisis , Prueba Serológica para COVID-19/métodos , COVID-19/inmunología , Imagen Óptica/métodos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas contra la COVID-19/inmunología , Humanos , Pruebas de Neutralización , Virus de la Estomatitis Vesicular Indiana/inmunologíaRESUMEN
Rationale: Hosts defend against viral infection by sensing viral pathogen-associated molecular patterns and activating antiviral innate immunity through TBK1-IRF3 signaling. However, the underlying molecular mechanism remains unclear. Methods: SiRNAs targeting Sirt1-7 were transfected into macrophages to screen the antiviral function. Sirt5 deficient mice or macrophages were subjected to viral infection to assess in vivo and in vitro function of Sirt5 by detecting cytokines, viral replicates and survival rate. Immunoprecipitation, WesternBlot and luciferase reporter assay were used to reveal molecular mechanism. Results: In this study, we functionally screened seven Sirtuin family members, and found that Sirtuin5 (Sirt5) promotes antiviral signaling and responses. Sirt5 deficiency leads to attenuated antiviral innate immunity in vivo and in vitro upon viral infection by decreasing TBK1-IRF3 activation and type I IFN production. Sirt5 overexpression increased antiviral innate immunity. Mechanism investigation revealed that Sirt5 interacts with DDX3 and demalonylates DDX3, which is critical for TBK1-IRF3 activation. Mutation of the demalonylation lysine sites (K66, K130, and K162) of DDX3 increased ifnß transcription. Furthermore, the acetylation on lysine 118 of DDX3 positively regulated ifnß transcription, whereas Sirt5 could not deacetylate this site. Conclusion: Sirt5 promotes anti- RNA and DNA virus innate immune responses by increasing TBK1 signaling through demalonylating DDX3, which identifies a novel regulatory pathway of antiviral innate immune response.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Inmunidad Innata , Macrófagos/inmunología , Sirtuinas/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Animales , ARN Helicasas DEAD-box/genética , Células HEK293 , Humanos , Lipoilación/genética , Lipoilación/inmunología , Macrófagos/virología , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Células RAW 264.7 , Sirtuinas/genética , Estomatitis Vesicular/genética , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
ABSTRACTThe recent impact of Ebola virus disease (EVD) on public health in Africa clearly demonstrates the need for a safe and efficacious vaccine to control outbreaks and mitigate its threat to global health. ERVEBO® is an effective recombinant Vesicular Stomatitis Virus (VSV)-vectored Ebola virus vaccine (VSV-EBOV) that was approved by the FDA and EMA in late 2019 for use in prevention of EVD. Since the parental virus VSV, which was used to construct VSV-EBOV, is pathogenic for livestock and the vaccine virus may be shed at low levels by vaccinated humans, widespread deployment of the vaccine requires investigation into its infectivity and transmissibility in VSV-susceptible livestock species. We therefore performed a comprehensive clinical analysis of the VSV-EBOV vaccine virus in swine to determine its infectivity and potential for transmission. A high dose of VSV-EBOV resulted in VSV-like clinical signs in swine, with a proportion of pigs developing ulcerative vesicular lesions at the nasal injection site and feet. Uninoculated contact control pigs co-mingled with VSV-EBOV-inoculated pigs did not become infected or display any clinical signs of disease, indicating the vaccine is not readily transmissible to naïve pigs during prolonged close contact. In contrast, virulent wild-type VSV Indiana had a shorter incubation period and was transmitted to contact control pigs. These results indicate that the VSV-EBOV vaccine causes vesicular illness in swine when administered at a high dose. Moreover, the study demonstrates the VSV-EBOV vaccine is not readily transmitted to uninfected pigs, encouraging its safe use as an effective human vaccine.
Asunto(s)
Vacunas contra el Virus del Ébola/efectos adversos , Vacunas contra el Virus del Ébola/inmunología , Ebolavirus/inmunología , Estomatitis Vesicular/transmisión , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/inmunología , Vesiculovirus/inmunología , África , Animales , Chlorocebus aethiops , Ebolavirus/genética , Femenino , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/virología , Humanos , Masculino , Modelos Animales , ARN Viral , Porcinos , Vacunación/métodos , Vacunas Sintéticas/efectos adversos , Vacunas Sintéticas/inmunología , Células Vero , Vesiculovirus/genéticaRESUMEN
In our clinical trials of oncolytic vesicular stomatitis virus expressing interferon beta (VSV-IFNß), several patients achieved initial responses followed by aggressive relapse. We show here that VSV-IFNß-escape tumors predictably express a point-mutated CSDE1P5S form of the RNA-binding Cold Shock Domain-containing E1 protein, which promotes escape as an inhibitor of VSV replication by disrupting viral transcription. Given time, VSV-IFNß evolves a compensatory mutation in the P/M Inter-Genic Region which rescues replication in CSDE1P5S cells. These data show that CSDE1 is a major cellular co-factor for VSV replication. However, CSDE1P5S also generates a neo-epitope recognized by non-tolerized T cells. We exploit this predictable neo-antigenesis to drive, and trap, tumors into an escape phenotype, which can be ambushed by vaccination against CSDE1P5S, preventing tumor escape. Combining frontline therapy with escape-targeting immunotherapy will be applicable across multiple therapies which drive tumor mutation/evolution and simultaneously generate novel, targetable immunopeptidomes associated with acquired treatment resistance.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Interferón beta/inmunología , Viroterapia Oncolítica/métodos , Virus Oncolíticos/inmunología , Proteínas de Unión al ARN/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Replicación Viral/inmunología , Animales , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Inmunoterapia/métodos , Interferón beta/metabolismo , Ratones Endogámicos C57BL , Mutación , Virus Oncolíticos/metabolismo , Virus Oncolíticos/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
The human complement system is an important part of the innate immune system. Its effector pathways largely mediate virus neutralization. Vesicular stomatitis virus (VSV) activates the classical pathway of the complement, leading to virus neutralization by lysis. Two host-derived membrane-associated regulators of complement activation (RCA), CD55 and CD46, which are incorporated into the VSV envelope during egress, confer protection by delaying/resisting complement-mediated neutralization. We showed previously that CD55 is more effective than CD46 in the inhibition of neutralization. In this study, we identified that, at the protein level, VSV infection resulted in the down-regulation of CD46 but not CD55. The mRNA of both the RCAs was significantly down-regulated by VSV, but it was delayed in the case of CD55. The immunoblot analysis of the levels of RCAs in the progeny virion harvested at three specific time intervals, points to an equal ratio of its distribution relative to viral proteins. Besides reconfirming the dominant role of CD55 over CD46 in shielding VSV from complement, our results also highlight the importance of the subtle modulation in the expression pattern of RCAs in a system naturally expressing them.
Asunto(s)
Antígenos CD55/inmunología , Proteínas del Sistema Complemento/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Células A549 , Línea Celular Tumoral , Activación de Complemento/inmunología , Células HeLa , Humanos , Proteína Cofactora de Membrana/inmunología , Pruebas de Neutralización/métodos , ARN Mensajero/inmunología , Virión/inmunologíaRESUMEN
BACKGROUND: Vesicular stomatitis (VS) is an acute, highly contagious and economically important zoonotic disease caused by the vesicular stomatitis virus (VSV). There is a need for effective and safe stable recombinant vaccine for the control of the disease. The human type 5 replication-defective adenovirus expression vector is a good way to construct recombinant vaccines. RESULTS: Three recombinant adenoviruses (rAd) were successfully constructed that expressed the VSV Indiana serotype glycoprotein (VSV-IN-G), VSV New Jersey serotype glycoprotein (VSV-NJ-G), and the G fusion protein (both serotypes of G [VSV-IN-G-NJ-G]) with potentiality to induce protective immunity. G proteins were successfully expressed with good immunogenicity. The rAds could induce the production of VSV antibodies in mice, and VSV neutralizing antibodies in goats, respectively. The neutralizing antibody titers could reach 1:32 in mice and 1:64 in goats. The rAds induced strong lymphocyte proliferation in mice and goats, which was significantly higher compared to the negative control groups. CONCLUSIONS: The three rAds constructed in the study expressed VSV-G proteins and induced both humoral and cellular immune responses in mice and goats. These results lay the foundation for further studies on the use of rAds in vaccines expressing VSV-G.