RESUMEN
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.
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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
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.
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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
The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.
Asunto(s)
Prolina , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Desarrollo de Vacunas , Estomatitis Vesicular , Vacunas Virales , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/genética , COVID-19/inmunología , COVID-19/prevención & control , COVID-19/virología , Vacunas contra la COVID-19/inmunología , Cricetinae , Humanos , Ratones , Prolina/inmunología , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunología , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/prevención & control , Estomatitis Vesicular/virología , Vesiculovirus/inmunología , Proteínas Virales/inmunología , Vacunas Virales/inmunologíaRESUMEN
The ongoing pandemic of coronavirus (CoV) disease 2019 (COVID-19) continues to exert a significant burden on health care systems worldwide. With limited treatments available, vaccination remains an effective strategy to counter transmission of severe acute respiratory syndrome CoV 2 (SARS-CoV-2). Recent discussions concerning vaccination strategies have focused on identifying vaccine platforms, number of doses, route of administration, and time to reach peak immunity against SARS-CoV-2. Here, we generated a single-dose, fast-acting vesicular stomatitis virus (VSV)-based vaccine derived from the licensed Ebola virus (EBOV) vaccine rVSV-ZEBOV, expressing the SARS-CoV-2 spike protein and the EBOV glycoprotein (VSV-SARS2-EBOV). Rhesus macaques vaccinated intramuscularly (i.m.) with a single dose of VSV-SARS2-EBOV were protected within 10 days and did not show signs of COVID-19 pneumonia. In contrast, intranasal (i.n.) vaccination resulted in limited immunogenicity and enhanced COVID-19 pneumonia compared to results for control animals. While both i.m. and i.n. vaccination induced neutralizing antibody titers, only i.m. vaccination resulted in a significant cellular immune response. RNA sequencing data bolstered these results by revealing robust activation of the innate and adaptive immune transcriptional signatures in the lungs of i.m. vaccinated animals only. Overall, the data demonstrate that VSV-SARS2-EBOV is a potent single-dose COVID-19 vaccine candidate that offers rapid protection based on the protective efficacy observed in our study. IMPORTANCE The vesicular stomatitis virus (VSV) vaccine platform rose to fame in 2019, when a VSV-based Ebola virus (EBOV) vaccine was approved by the European Medicines Agency and the U.S. Food and Drug Administration for human use against the deadly disease. Here, we demonstrate the protective efficacy of a VSV-EBOV-based COVID-19 vaccine against challenge in nonhuman primates (NHPs). When a single dose of the VSV-SARS2-EBOV vaccine was administered intramuscularly (i.m.), the NHPs were protected from COVID-19 within 10 days. In contrast, if the vaccine was administered intranasally, there was no benefit from the vaccine and the NHPs developed pneumonia. The i.m. vaccinated NHPs quickly developed antigen-specific IgG, including neutralizing antibodies. Transcriptional analysis highlighted the development of protective innate and adaptive immune responses in the i.m. vaccination group only.
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Vacunas contra la COVID-19 , COVID-19 , Vacunas contra el Virus del Ébola , Ebolavirus , Macaca mulatta , Estomatitis Vesicular , Animales , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/administración & dosificación , Vacunas contra la COVID-19/uso terapéutico , Vacunas contra el Virus del Ébola/genética , Vacunas contra el Virus del Ébola/inmunología , Vacunas contra el Virus del Ébola/uso terapéutico , Ebolavirus/genética , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/genética , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Macaca mulatta/inmunología , SARS-CoV-2 , Vacunación/métodos , Estomatitis Vesicular/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/prevención & control , Vesiculovirus/genéticaRESUMEN
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
The antiviral innate immunity is the first line of host defense against viral infection. Mitochondrial antiviral signaling protein (MAVS, also named Cardif/IPS-1/VISA) is a critical protein in RNA virus-induced antiviral signaling pathways. Our previous research suggested that E3 ubiquitin-protein ligases RING-finger protein (RNF90) negatively regulate cellular antiviral responses by targeting STING for degradation, though its role in RNA virus infection remains unknown. This study demonstrated that RNF90 negatively regulated RNA virus-triggered antiviral innate immune responses in RNF90-silenced PMA-THP1 cells, RNF90-deficient cells (including HaCaTs, MEFs, and BMDMs), and RNF90-deficient mice. However, RNF90 regulated RNA virus-triggered antiviral innate immune responses independent of STING. RNF90 promoted K48-linked ubiquitination of MAVS and its proteasome-dependent degradation, leading to the inhibition of innate immune responses. Altogether, our findings suggested a novel function and mechanism of RNF90 in antiviral innate immunity.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inmunidad Innata , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Estomatitis Vesicular/metabolismo , Vesiculovirus/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Chlorocebus aethiops , Citocinas/genética , Citocinas/inmunología , Citocinas/metabolismo , Células HEK293 , Células HaCaT , Interacciones Huésped-Patógeno , Humanos , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Transducción de Señal , Células THP-1 , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/inmunología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitinación , Células Vero , Estomatitis Vesicular/genética , Estomatitis Vesicular/inmunología , Estomatitis Vesicular/virología , Vesiculovirus/patogenicidadRESUMEN
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
Interferons induce cell-intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. Using vesicular stomatitis virus (VSV), we report that the host cell N6-adenosine messenger RNA (mRNA) cap methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m7Gpppm6Am and define the substrate requirements for this modification. Functional assays revealed that the PCIF1-dependent modification of VSV mRNA cap structures is inert with regard to mRNA stability, translation, and viral infectivity but attenuates the antiviral effects of the treatment of cells with interferon-ß. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-ß treatment. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m7Gpppm6Am This work identifies a function of PCIF1 and cap-proximal m6Am in attenuation of the host response to VSV infection that likely extends to other viruses.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Interferón beta/inmunología , Proteínas Nucleares/metabolismo , Caperuzas de ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Interacciones Huésped-Patógeno , Humanos , Interferón beta/genética , Metilación , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Caperuzas de ARN/genética , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genética , Estomatitis Vesicular/genética , Estomatitis Vesicular/metabolismo , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/química , Virus de la Estomatitis Vesicular Indiana/genética , Replicación ViralRESUMEN
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.
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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
BACKGROUND: The effect of malaria infection on the immunogenicity of the recombinant vesicular stomatitis virus-Zaire Ebola virus envelope glycoprotein (GP) vaccine (rVSVΔG-ZEBOV-GP) (ERVEBO) is unknown. METHODS: The Sierra Leone Trial to Introduce a Vaccine Against Ebola (STRIVE) vaccinated 7998 asymptomatic adults with rVSVΔG-ZEBOV-GP during the 2014-2016 Ebola epidemic. In STRIVE's immunogenicity substudy, participants provided blood samples at baseline and at 1, 6, and 9-12 months. Anti-GP binding and neutralizing antibodies were measured using validated assays. Baseline samples were tested for malaria parasites by polymerase chain reaction. RESULTS: Overall, 506 participants enrolled in the immunogenicity substudy and had ≥1 postvaccination antibody titer. Of 499 participants with a result, baseline malaria parasitemia was detected in 73 (14.6%). All GP enzyme-linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT) geometric mean titers (GMTs) at 1, 6, and 9-12 months were above baseline, and 94.1% of participants showed seroresponse by GP-ELISA (≥2-fold rise and ≥200 ELISA units/mL), while 81.5% showed seroresponse by PRNT (≥4-fold rise) at ≥1 postvaccination assessment. In participants with baseline malaria parasitemia, the PRNT seroresponse proportion was lower, while PRNT GMTs and GP-ELISA seroresponse and GMTs showed a trend toward lower responses at 6 and 9-12 months. CONCLUSION: Asymptomatic adults with or without malaria parasitemia had robust immune responses to rVSVΔG-ZEBOV-GP, persisting for 9-12 months. Responses in those with malaria parasitemia were somewhat lower.
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Vacunas contra el Virus del Ébola/inmunología , Ebolavirus , Fiebre Hemorrágica Ebola/prevención & control , Inmunogenicidad Vacunal , Estomatitis Vesicular/inmunología , Proteínas del Envoltorio Viral/inmunología , Adolescente , Adulto , Anciano , Animales , Anticuerpos Antivirales/sangre , Infecciones Asintomáticas , Vacunas contra el Virus del Ébola/administración & dosificación , Vacunas contra el Virus del Ébola/efectos adversos , Ebolavirus/genética , Ebolavirus/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Femenino , Fiebre Hemorrágica Ebola/inmunología , Humanos , Malaria , Masculino , Persona de Mediana Edad , Parasitemia/prevención & control , Proteínas Recombinantes , Sierra Leona , Proteínas del Envoltorio Viral/efectos adversosRESUMEN
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.
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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.
Asunto(s)
Glicoproteínas de Membrana/inmunología , Estomatitis Vesicular/prevención & control , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular New Jersey/inmunología , Proteínas del Envoltorio Viral/inmunología , Adenoviridae , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Femenino , Enfermedades de las Cabras/inmunología , Enfermedades de las Cabras/prevención & control , Enfermedades de las Cabras/virología , Cabras , Inmunidad Celular , Inmunidad Humoral , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones Endogámicos BALB C , Vacunas Sintéticas/inmunología , Estomatitis Vesicular/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/inmunologíaRESUMEN
Neutrophils are innate leukocytes that mount a rapid response to invading pathogens and sites of inflammation. Although neutrophils were traditionally considered responders to bacterial infections, recent advances have demonstrated that they are interconnected with both viral infections and cancers. One promising treatment strategy for cancers is to administer an oncolytic virus to activate the immune system and directly lyse cancerous cells. A detailed characterization of how the innate immune system responds to a viral-based therapy is paramount in identifying its systemic effects. This study analyzed how administering the rhabdovirus vesicular stomatitis virus (VSV) intravenously at 1 × 109 PFU acutely influenced neutrophil populations. Bone marrow, blood, lungs, and spleen were acquired three- and 24-h after administration of VSV for analysis of neutrophils by flow cytometry. Infection with VSV caused neutrophils to rapidly egress from the bone marrow and accumulate in the lungs. A dramatic increase in immature neutrophils was observed in the lungs, as was an increase in the antigen presentation potential of these cells within the spleen. Furthermore, the potential for neutrophils to acquire viral transgene-encoded proteins was monitored using a variant of VSV that expressed enhanced green fluorescent protein (GFP). If an in vitro population of splenocytes were exposed to αCD3 and αCD28, a substantial proportion of the neutrophils would become GFP-positive. This suggested that the neutrophils could either acquire more virus-encoded antigens from infected splenocytes or were being directly infected. Five different dosing regimens were tested in mice, and it was determined that a single dose of VSV or two doses of VSV administered at a 24-h interval, resulted in a substantial proportion of neutrophils in the bone marrow becoming GFP-positive. This correlated with a decrease in the number of splenic neutrophils. Two doses administered at intervals longer than 24-h did not have these effects, suggesting that neutrophils became resistant to antigen uptake or direct infection with VSV beyond 24-h of activation. These findings implicated neutrophils as major contributors to oncolytic rhabdoviral therapies. They also provide several clear future directions for research and suggest that neutrophils should be carefully monitored during the development of all oncolytic virus-based treatment regimens.
Asunto(s)
Presentación de Antígeno/inmunología , Neutrófilos/inmunología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas no Estructurales Virales/metabolismo , Animales , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Estomatitis Vesicular/terapia , Estomatitis Vesicular/virología , Proteínas no Estructurales Virales/inmunologíaRESUMEN
The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22-20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22-20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein.
Asunto(s)
Interferón beta/inmunología , FN-kappa B/inmunología , Estomatitis Vesicular/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Línea Celular , Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Interferón beta/genética , Ratones , FN-kappa B/genética , Estomatitis Vesicular/genética , Estomatitis Vesicular/virología , Virus de la Estomatitis Vesicular Indiana/genética , Virus de la Estomatitis Vesicular Indiana/fisiología , Proteínas de la Matriz Viral/genéticaRESUMEN
The neuroepithelium is a nasal barrier surface populated by olfactory sensory neurons that detect odorants in the airway and convey this information directly to the brain via axon fibers. This barrier surface is especially vulnerable to infection, yet respiratory infections rarely cause fatal encephalitis, suggesting a highly evolved immunological defense. Here, using a mouse model, we sought to understand the mechanism by which innate and adaptive immune cells thwart neuroinvasion by vesicular stomatitis virus (VSV), a potentially lethal virus that uses olfactory sensory neurons to enter the brain after nasal infection. Fate-mapping studies demonstrated that infected central nervous system (CNS) neurons were cleared noncytolytically, yet specific deletion of major histocompatibility complex class I (MHC I) from these neurons unexpectedly had no effect on viral control. Intravital imaging studies of calcium signaling in virus-specific CD8+ T cells revealed instead that brain-resident microglia were the relevant source of viral peptide-MHC I complexes. Microglia were not infected by the virus but were found to cross-present antigen after acquisition from adjacent neurons. Microglia depletion interfered with T cell calcium signaling and antiviral control in the brain after nasal infection. Collectively, these data demonstrate that microglia provide a front-line defense against a neuroinvasive nasal infection by cross-presenting antigen to antiviral T cells that noncytolytically cleanse neurons. Disruptions in this innate defense likely render the brain susceptible to neurotropic viruses like VSV that attempt to enter the CNS via the nose.
Asunto(s)
Presentación de Antígeno/inmunología , Encéfalo/inmunología , Linfocitos T CD8-positivos/inmunología , Microglía/inmunología , Células Neuroepiteliales/inmunología , Nariz/virología , Estomatitis Vesicular/inmunología , Animales , Encéfalo/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microglía/virología , Células Neuroepiteliales/virología , Estomatitis Vesicular/virologíaRESUMEN
Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor ß (TGF-ß). Lack of Mertk in Mertk-/- mice prevents induction of IL-10 and TGF-ß, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV.