Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Cell ; 182(5): 1271-1283.e16, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32795413

RESUMEN

There is an urgent need for vaccines against coronavirus disease 2019 (COVID-19) because of the ongoing SARS-CoV-2 pandemic. Among all approaches, a messenger RNA (mRNA)-based vaccine has emerged as a rapid and versatile platform to quickly respond to this challenge. Here, we developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor binding domain (RBD) of SARS-CoV-2 as a vaccine candidate (called ARCoV). Intramuscular immunization of ARCoV mRNA-LNP elicited robust neutralizing antibodies against SARS-CoV-2 as well as a Th1-biased cellular response in mice and non-human primates. Two doses of ARCoV immunization in mice conferred complete protection against the challenge of a SARS-CoV-2 mouse-adapted strain. Additionally, ARCoV is manufactured as a liquid formulation and can be stored at room temperature for at least 1 week. ARCoV is currently being evaluated in phase 1 clinical trials.


Asunto(s)
ARN Mensajero/genética , ARN Viral/genética , Vacunas Sintéticas/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Sitios de Unión , Vacunas contra la COVID-19 , Chlorocebus aethiops , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Femenino , Células HEK293 , Células HeLa , Humanos , Inmunogenicidad Vacunal , Inyecciones Intramusculares , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos ICR , Nanopartículas/química , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células TH1/inmunología , Potencia de la Vacuna , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Células Vero , Vacunas Virales/administración & dosificación , Vacunas Virales/genética
2.
Signal Transduct Target Ther ; 6(1): 438, 2021 12 24.
Artículo en Inglés | MEDLINE | ID: mdl-34952914

RESUMEN

Messenger RNA (mRNA) vaccine technology has shown its power in preventing the ongoing COVID-19 pandemic. Two mRNA vaccines targeting the full-length S protein of SARS-CoV-2 have been authorized for emergency use. Recently, we have developed a lipid nanoparticle-encapsulated mRNA (mRNA-LNP) encoding the receptor-binding domain (RBD) of SARS-CoV-2 (termed ARCoV), which confers complete protection in mouse model. Herein, we further characterized the protection efficacy of ARCoV in nonhuman primates and the long-term stability under normal refrigerator temperature. Intramuscular immunization of two doses of ARCoV elicited robust neutralizing antibodies as well as cellular response against SARS-CoV-2 in cynomolgus macaques. More importantly, ARCoV vaccination in macaques significantly protected animals from acute lung lesions caused by SARS-CoV-2, and viral replication in lungs and secretion in nasal swabs were completely cleared in all animals immunized with low or high doses of ARCoV. No evidence of antibody-dependent enhancement of infection was observed throughout the study. Finally, extensive stability assays showed that ARCoV can be stored at 2-8 °C for at least 6 months without decrease of immunogenicity. All these promising results strongly support the ongoing clinical trial.


Asunto(s)
Vacunas contra la COVID-19/farmacología , COVID-19/inmunología , Inmunogenicidad Vacunal , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas de ARNm/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Chlorocebus aethiops , Humanos , Macaca fascicularis , Células Vero , Vacunas de ARNm/inmunología
3.
Cell Host Microbe ; 28(1): 124-133.e4, 2020 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-32485164

RESUMEN

Since December 2019, a novel coronavirus SARS-CoV-2 has emerged and rapidly spread throughout the world, resulting in a global public health emergency. The lack of vaccine and antivirals has brought an urgent need for an animal model. Human angiotensin-converting enzyme II (ACE2) has been identified as a functional receptor for SARS-CoV-2. In this study, we generated a mouse model expressing human ACE2 (hACE2) by using CRISPR/Cas9 knockin technology. In comparison with wild-type C57BL/6 mice, both young and aged hACE2 mice sustained high viral loads in lung, trachea, and brain upon intranasal infection. Although fatalities were not observed, interstitial pneumonia and elevated cytokines were seen in SARS-CoV-2 infected-aged hACE2 mice. Interestingly, intragastric inoculation of SARS-CoV-2 was seen to cause productive infection and lead to pulmonary pathological changes in hACE2 mice. Overall, this animal model described here provides a useful tool for studying SARS-CoV-2 transmission and pathogenesis and evaluating COVID-19 vaccines and therapeutics.


Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Pandemias , Neumonía Viral , Envejecimiento , Enzima Convertidora de Angiotensina 2 , Animales , Encéfalo/virología , COVID-19 , Sistemas CRISPR-Cas , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Citocinas/sangre , Técnicas de Sustitución del Gen , Pulmón/patología , Pulmón/virología , Enfermedades Pulmonares Intersticiales/patología , Nariz/virología , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , ARN Viral/análisis , SARS-CoV-2 , Estómago/virología , Tráquea/virología , Carga Viral , Replicación Viral
4.
EBioMedicine ; 12: 170-177, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27693104

RESUMEN

Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV). Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.


Asunto(s)
Infección por el Virus Zika/virología , Virus Zika/fisiología , Animales , Línea Celular , Modelos Animales de Enfermedad , Fiebre , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunohistoquímica , Macaca mulatta , Reacción en Cadena de la Polimerasa , Primates , ARN Viral , Tropismo Viral , Viremia/virología , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/inmunología
5.
mBio ; 6(2)2015 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-25805734

RESUMEN

UNLABELLED: Hepatic injuries in hepatitis B virus (HBV) patients are caused by immune responses of the host. In our previous study, microRNA-146a (miR-146a), an innate immunity-related miRNA, and complement factor H (CFH), an important negative regulator of the alternative pathway of complement activation, were differentially expressed in HBV-expressing and HBV-free hepatocytes. Here, the roles of these factors in HBV-related liver inflammation were analyzed in detail. The expression levels of miR-146a and CFH in HBV-expressing hepatocytes were assessed via analyses of hepatocyte cell lines, transgenic mice, adenovirus-infected mice, and HBV-positive human liver samples. The expression level of miR-146a was upregulated in HBV-expressing Huh-7 hepatocytes, HBV-expressing mice, and patients with HBV infection. Further results demonstrated that the HBV X protein (HBx) was responsible for its effects on miR-146a expression through NF-κB-mediated enhancement of miR-146a promoter activity. HBV/HBx also downregulated the expression of CFH mRNA in hepatocyte cell lines and the livers of humans and transgenic mice. Furthermore, overexpression and inhibition of miR-146a in Huh-7 cells downregulated and upregulated CFH mRNA levels, respectively. Luciferase reporter assays demonstrated that miR-146a downregulated CFH mRNA expression in hepatocytes via 3'-untranslated-region (UTR) pairing. The overall effect of this process in vivo is to promote liver inflammation. These results demonstrate that the HBx-miR-146a-CFH-complement activation regulation pathway might play an important role in the immunopathogenesis of chronic HBV infection. These findings have important implications for understanding the immunopathogenesis of chronic hepatitis B and developing effective therapeutic interventions. IMPORTANCE: Hepatitis B virus (HBV) remains an important pathogen and can cause severe liver diseases, including hepatitis, liver cirrhosis, and hepatocellular carcinoma. Although HBV was found in 1966, the molecular mechanisms of pathogenesis are still poorly understood. In the present study, we found that the HBV X protein (HBx) promoted the expression of miR-146a, an innate immunity-related miRNA, through the NF-κB signal pathway and that increasingly expressed miR-146a downregulated its target complement factor H (CFH), an important negative regulator of the complement alternative pathway, leading to the promotion of liver inflammation. We demonstrated that the HBx-miR-146a-CFH-complement activation regulation pathway is potentially an important mechanism of immunopathogenesis caused by chronic HBV infection. Our data provide a novel molecular mechanism of HBV pathogenesis and thus help to understand the correlations between the complement system, an important part of innate immunity, and HBV-associated disease. These findings will also be important to identify potential therapeutic targets for HBV infection.


Asunto(s)
Factor H de Complemento/antagonistas & inhibidores , Hepatitis B/inmunología , Hepatitis B/patología , Interacciones Huésped-Patógeno , MicroARNs/biosíntesis , Transactivadores/metabolismo , Regulación hacia Arriba , Animales , Línea Celular , Hepatocitos/virología , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Reguladoras y Accesorias Virales
6.
World J Gastroenterol ; 18(16): 1892-902, 2012 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-22563169

RESUMEN

AIM: To investigate the relationship between over-expression of urokinase plasminogen activator (uPA) and hepatitis B virus (HBV) related liver diseases in a transgenic mouse model. METHODS: Albumin-tetracycline reverse transcriptional activator and tetO-uPA transgenic mice were generated respectively through pronuclear injection and crossed to produce the double transgenic in-alb-uPA mice, for which doxycycline (Dox)-inducible and liver-specific over-expression of uPA can be achieved. Hydrodynamic transfection of plasmid adeno-associated virus (AAV)-1.3 HBV was performed through the tail veins of the Dox-induced in-alb-uPA mice. Expression of uPA and HBV antigens were analyzed through double-staining immunohistochemical assay. Cytokine production was detected by enzyme linked immunosorbent assay and α-fetoprotein (AFP) mRNA level was evaluated through real-time quantitative polymerase chain reaction. RESULTS: Plasmid AAV-1.3 HBV hydrodynamic transfection in Dox-induced transgenic mice not only resulted in severe liver injury with hepatocarcinoma-like histological changes and hepatic AFP production, but also showed an increased serum level of HBV antigens and cytokines like interleukin-6 and tumor necrosis factor-α, compared with the control group. CONCLUSION: Over-expression of uPA plays a synergistic role in the development of liver injury, inflammation and regeneration during acute HBV infection.


Asunto(s)
Dependovirus/genética , Virus de la Hepatitis B/genética , Hepatopatías/etiología , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Animales , Citocinas/biosíntesis , Doxorrubicina/farmacología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Riesgo , Transfección , alfa-Fetoproteínas/genética
7.
Bing Du Xue Bao ; 26(1): 20-6, 2010 Jan.
Artículo en Zh | MEDLINE | ID: mdl-20329554

RESUMEN

To develop a HBV infection mouse model by hydrodynamic-based transfection and further to optimize the method of development of HBV infection mouse model. We first developed a construct which contained inverted terminal repeat elements (ITR) of adeno-associated virus (AAV) and 1. 3 copies of HBV genome (ayw subtype). The pAAV-HBV1. 3 DNA was then injected hydrodynamically into the tail veins of C57BL/6 mice in 5 seconds. The virus load in serum and liver was assayed by ELISA and Real-time PCR. The expression of virus antigen and the pathologic changes of liver were analyzed by HE and immunohistochemical staining. Meanwhile, to develop HBV transfected immunosuppressied mouse, mice were injected intraperitoneally triple with 0.2 ml dexamethason (50 mg/kg) every two days before HBV transfection. The levels of HBsAg and HBeAg were assayed by ELISA. Our data showed: (1) HBsAg and HBeAg were positive (100%) in serum and liver of experimental normal mouse at day 10 after HBV transfection, and became negative at day 30 and day 60. Meanwhile the viral load in serum and liver in experimental group was significantly higher than that in control group at day 10, 30 and 60 after HBV transfection (P < 0.01, P < 0.05, respectively). (2) HBsAg and HBeAg in serum in immunosuppressed mouse model were positive until 60 days. In conclusion, a HBV infection mouse model was developed successfully by hydrodynamic-based transfection. By suppressing the immune status of mice injected with dexamethasone, the expression of HBV antigens was extended longer than that in normal adult mice. These models pave a way for HBV research and evaluation of HBV vaccine and drug development.


Asunto(s)
Dexametasona/administración & dosificación , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica/efectos de los fármacos , Antígenos de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B/virología , Inmunosupresores/administración & dosificación , Transfección/métodos , Animales , Dependovirus/genética , Dependovirus/metabolismo , Dexametasona/inmunología , Femenino , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Hepatitis B/inmunología , Antígenos de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Humanos , Inmunosupresores/inmunología , Hígado/inmunología , Hígado/virología , Ratones , Ratones Endogámicos C57BL
8.
Sheng Wu Gong Cheng Xue Bao ; 23(2): 223-8, 2007 Mar.
Artículo en Zh | MEDLINE | ID: mdl-17460892

RESUMEN

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.


Asunto(s)
Fibroblastos/metabolismo , Albúmina Sérica/genética , Transfección/métodos , Animales , Secuencia de Bases , Southern Blotting , Supervivencia Celular/genética , Células Cultivadas , Clonación Molecular , Electroporación , Feto , Fibroblastos/citología , Humanos , Cariotipificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Porcinos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA