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1.
Sci Transl Med ; 13(597)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108251

RESUMO

A safe and protective Lassa virus vaccine is crucially needed in Western Africa to stem the recurrent outbreaks of Lassa virus infections in Nigeria and the emergence of Lassa virus in previously unaffected countries, such as Benin and Togo. Major challenges in developing a Lassa virus vaccine include the high diversity of circulating strains and their reemergence from 1 year to another. To address each of these challenges, we immunized cynomolgus monkeys with a measles virus vector expressing the Lassa virus glycoprotein and nucleoprotein of the prototypic Lassa virus strain Josiah (MeV-NP). To evaluate vaccine efficacy against heterologous strains of Lassa virus, we challenged the monkeys a month later with heterologous strains from lineage II or lineage VII, finding that the vaccine was protective against these strains. A second cohort of monkeys was challenged 1 year later with the homologous Josiah strain, finding that a single dose of MeV-NP was sufficient to protect all vaccinated monkeys. These studies demonstrate that MeV-NP can generate both long-lasting immune responses and responses that are able to protect against diverse strains of Lassa virus.

2.
Mol Ecol Resour ; 21(5): 1620-1640, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33586292

RESUMO

To salvage marine ecosystems from fishery overexploitation, sustainable and efficient aquaculture must be emphasized. The knowledge obtained from available genome sequence of marine organisms has accelerated marine aquaculture in many cases. The black tiger shrimp (Penaeus monodon) is one of the most prominent cultured penaeid shrimps (Crustacean) with an average annual global production of half a million tons in the last decade. However, its currently available genome assemblies lack the contiguity and completeness required for accurate genome annotation due to the highly repetitive nature of the genome and technical difficulty in extracting high-quality, high-molecular weight DNA. Here, we report the first chromosome-level whole-genome assembly of P. monodon. The combination of long-read Pacific Biosciences (PacBio) and long-range Chicago and Hi-C technologies enabled a successful assembly of this first high-quality genome sequence. The final assembly covered 2.39 Gb (92.3% of the estimated genome size) and contained 44 pseudomolecules, corresponding to the haploid chromosome number. Repetitive elements occupied a substantial portion of the assembly (62.5%), the highest of the figures reported among crustacean species. The availability of this high-quality genome assembly enabled the identification of genes associated with rapid growth in the black tiger shrimp through the comparison of hepatopancreas transcriptome of slow-growing and fast-growing shrimps. The results highlighted several growth-associated genes. Our high-quality genome assembly provides an invaluable resource for genetic improvement and breeding penaeid shrimp in aquaculture. The availability of P. monodon genome enables analyses of ecological impact, environment adaptation and evolution, as well as the role of the genome to protect the ecological resources by promoting sustainable shrimp farming.

3.
RNA Biol ; 18(5): 732-744, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33459164

RESUMO

Flaviviruses such as the dengue (DENV) and the Zika virus (ZIKV) are important human pathogens causing around 100 million symptomatic infections each year. During infection, small subgenomic flavivirus RNAs (sfRNAs) are formed inside the infected host cell as a result of incomplete degradation of the viral RNA genome by cellular exoribonuclease XRN1. Although the full extent of sfRNA functions is to be revealed, these non-coding RNAs are key virulence factors and their detrimental effects on multiple cellular processes seem to consistently involve molecular interactions with RNA-binding proteins (RBPs). Discovery of such sfRNA-binding host-factors has followed established biochemical pull-down approaches skewed towards highly abundant proteins hampering proteome-wide coverage. Yeast three-hybrid (Y3H) systems represent an attractive alternative approach. To facilitate proteome-wide screens for RBP, we revisited and improved existing RNA-Y3H methodology by (1) implementing full-length ORF libraries in combination with (2) efficient yeast mating to increase screening depth and sensitivity, and (3) stringent negative controls to eliminate over-representation of non-specific RNA-binders. These improvements were validated employing the well-characterized interaction between DDX6 (DEAD-box helicase 6) and sfRNA of DENV as paradigm. Our advanced Y3H system was used to screen for human proteins binding to DENV and ZIKV sfRNA, resulting in a list of 69 putative sfRNA-binders, including several previously reported as well as numerous novel RBP host factors. Our methodology requiring no sophisticated infrastructure or analytic pipeline may be employed for the discovery of meaningful RNA-protein interactions at large scale in other fields.

4.
Mol Cell Proteomics ; 20: 100049, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33515806

RESUMO

Viruses manipulate the central machineries of host cells to their advantage. They prevent host cell antiviral responses to create a favorable environment for their survival and propagation. Measles virus (MV) encodes two nonstructural proteins MV-V and MV-C known to counteract the host interferon response and to regulate cell death pathways. Several molecular mechanisms underlining MV-V regulation of innate immunity and cell death pathways have been proposed, whereas MV-C host-interacting proteins are less studied. We suggest that some cellular factors that are controlled by MV-C protein during viral replication could be components of innate immunity and the cell death pathways. To determine which host factors are targeted by MV-C, we captured both direct and indirect host-interacting proteins of MV-C protein. For this, we used a strategy based on recombinant viruses expressing tagged viral proteins followed by affinity purification and a bottom-up mass spectrometry analysis. From the list of host proteins specifically interacting with MV-C protein in different cell lines, we selected the host targets that belong to immunity and cell death pathways for further validation. Direct protein interaction partners of MV-C were determined by applying protein complementation assay and the bioluminescence resonance energy transfer approach. As a result, we found that MV-C protein specifically interacts with p65-iASPP protein complex that controls both cell death and innate immunity pathways and evaluated the significance of these host factors on virus replication.

5.
J Virol ; 94(22)2020 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-32878892

RESUMO

Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response.IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

7.
J Thorac Oncol ; 15(5): 827-842, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31945495

RESUMO

INTRODUCTION: Oncolytic immunotherapy is based on the use of nonpathogenic replicative oncolytic viruses that infect and kill tumor cells exclusively. Recently, we found that the spontaneous oncolytic activity of the Schwarz strain of measles virus (MV) against human malignant pleural mesothelioma (MPM) depends on defects in the antiviral type I interferon (IFN-I) response in tumor cells. METHODS: In this study, we studied three independent human MPM bio-collections to identify the defects in the IFN-I responses in tumor cells. RESULTS: We show that the most frequent defect is the homozygous deletions (HDs) of all the 14 IFN-I genes (IFN-α and IFN-ß) that we found in more than half of MV-sensitive MPM cell lines. These HDs occur together with the HDs of the tumor suppressor gene CDKN2A also located in the 9p21.3 chromosome region. Therefore, the IFN-I-/- MPM cell lines develop a partial and weak IFN-I response when they are exposed to the virus compared with that of normal cells and MV-resistant MPM cell lines. This response consists of the expression of a restricted number of IFN-stimulated genes that do not depend on the presence of IFN-I. In addition, the IFN-I-/- MPM cell lines infected by MV also develop a pro-inflammatory response associated with stress of the endoplasmic reticulum. CONCLUSION: Our study emphasizes the link between HDs of IFN-I encoding genes and the CDKN2A gene in MPM and sensitivity to MV oncolytic immunotherapy.

8.
Methods Mol Biol ; 2058: 127-132, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31486035

RESUMO

Oncolytic immunotherapy efficacy relies partially on the induction of immunogenic tumor cell death following infection with oncolytic viruses (OV) to induce an antitumor immune response. Here, we describe a method to determine if an OV is able to induce such an immunogenic tumor cell death. This method consists in testing whether tumor cells lysed by an OV are able to induce the maturation of human monocyte-derived immature dendritic cells (Mo-iDC).

9.
Sci Rep ; 9(1): 20134, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882812

RESUMO

T. capitatus is widely used in traditional medicine in Tunisia. The main goal of this study was to evaluate the phytochemical composition, the fatty acids profile, the antioxidant, antibacterial, and antifungal activities as well as the cytotoxic potential of the essential oil (EO) of this plant. The identification and the quantification of the different constituents of the tested EO was determined by gas chromatography-mass spectrometry (GC-MS). Antioxidant activities were evaluated by spectrophotometric methods and chemical tests. HCT 116 cells were used to evaluate the cytotoxic effect of the EO. The microdilution method was conducted to evaluate the antibacterial activity. Poisoned food method was used to test the antifungal activities against fungi species such Aspergillus niger and Aspergillus flavus. The EO presented several components, mainly monoterpenes. Results revealed that T. capitatus EO is not cytotoxic and showed excellent antioxidant activity with a dose dependent manner. Regarding antimicrobial activity, T. capitatus EO was efficient against all tested bacteria and fungi.


Assuntos
Ácidos Graxos/química , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Compostos Fitoquímicos/química , Extratos Vegetais/química , Thymus (Planta)/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Fungos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Óleos Voláteis/análise , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/farmacologia
10.
Cell Host Microbe ; 26(6): 823-835.e11, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31761719

RESUMO

RNA-binding proteins (RBPs) perform key cellular activities by controlling the function of bound RNAs. The widely held assumption that RBPs are strictly intracellular has been challenged by the discovery of secreted RBPs. However, extracellular RBPs have been described in eukaryotes, while secreted bacterial RBPs have not been reported. Here, we show that the bacterial pathogen Listeria monocytogenes secretes a small RBP that we named Zea. We show that Zea binds a subset of L. monocytogenes RNAs, causing their accumulation in the extracellular medium. Furthermore, during L. monocytogenes infection, Zea binds RIG-I, the non-self-RNA innate immunity sensor, potentiating interferon-ß production. Mouse infection studies reveal that Zea affects L. monocytogenes virulence. Together, our results unveil that bacterial RNAs can be present extracellularly in association with RBPs, acting as "social RNAs" to trigger a host response during infection.


Assuntos
Proteína DEAD-box 58/metabolismo , Listeria monocytogenes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Proteína DEAD-box 58/imunologia , Células HEK293 , Interações entre Hospedeiro e Microrganismos , Humanos , Imunidade Inata , Interferon beta/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Camundongos , RNA Bacteriano/metabolismo , Transdução de Sinais/imunologia , Virulência/imunologia
11.
Sci Transl Med ; 11(512)2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578242

RESUMO

Lassa fever is a major threat in Western Africa. The large number of people living at risk for this disease calls for the development of a vaccine against Lassa virus (LASV). We generated live-attenuated LASV vaccines based on measles virus and Mopeia virus platforms and expressing different LASV antigens, with the aim to develop a vaccine able to protect after a single shot. We compared the efficacy of these vaccines against LASV in cynomolgus monkeys. The vaccines were well tolerated and protected the animals from LASV infection and disease after a single immunization but with varying efficacy. Analysis of the immune responses showed that complete protection was associated with robust secondary T cell and antibody responses against LASV. Transcriptomic and proteomic analyses showed an early activation of innate immunity and T cell priming after immunization with the most effective vaccines, with changes detectable as early as 2 days after immunization. The most efficacious vaccine candidate, a measles vector simultaneously expressing LASV glycoprotein and nucleoprotein, has been selected for further clinical evaluation.


Assuntos
Glicoproteínas/imunologia , Nucleoproteínas/imunologia , Proteínas Virais/imunologia , Animais , Linhagem Celular , Citometria de Fluxo , Humanos , Febre Lassa/imunologia , Febre Lassa/prevenção & controle , Febre Lassa/virologia , Vírus Lassa , Macaca fascicularis , Masculino , Proteômica , Transcriptoma , Vacinação/métodos
12.
Sci Signal ; 12(601)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575732

RESUMO

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I- and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I-dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.


Assuntos
Antivirais/metabolismo , Proteína DEAD-box 58/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/genética , Enterovirus Humano B/genética , Enterovirus Humano B/fisiologia , Células HEK293 , Células HeLa , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Mengovirus/genética , Mengovirus/fisiologia , Ligação Proteica , RNA Helicases/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Células Vero
13.
PLoS Negl Trop Dis ; 13(8): e0007299, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31412040

RESUMO

BACKGROUND: To be transmitted to vertebrate hosts via the saliva of their vectors, arthropod-borne viruses have to cross several barriers in the mosquito body, including the midgut infection and escape barriers. Yellow fever virus (YFV) belongs to the genus Flavivirus, which includes human viruses transmitted by Aedes mosquitoes, such as dengue and Zika viruses. The live-attenuated YFV-17D vaccine has been used safely and efficiently on a large scale since the end of World War II. Early studies have shown, using viral titration from salivary glands of infected mosquitoes, that YFV-17D can infect Aedes aegypti midgut, but does not disseminate to other tissues. METHODOLOGY/PRINCIPAL FINDINGS: Here, we re-visited this issue using a panel of techniques, such as RT-qPCR, Western blot, immunofluorescence and titration assays. We showed that YFV-17D replication was not efficient in Aedes aegypti midgut, as compared to the clinical isolate YFV-Dakar. Viruses that replicated in the midgut failed to disseminate to secondary organs. When injected into the thorax of mosquitoes, viruses succeeded in replicating into midgut-associated tissues, suggesting that, during natural infection, the block for YFV-17D replication occurs at the basal membrane of the midgut. CONCLUSIONS/SIGNIFICANCE: The two barriers associated with Ae. aegypti midgut prevent YFV-17D replication. Our study contributes to our basic understanding of vector-pathogen interactions and may also aid in the development of non-transmissible live virus vaccines.


Assuntos
Aedes/virologia , Trato Gastrointestinal/virologia , Replicação Viral/efeitos dos fármacos , Vacina contra Febre Amarela/farmacologia , Vírus da Febre Amarela/efeitos dos fármacos , Vírus da Febre Amarela/crescimento & desenvolvimento , Animais , Linhagem Celular , Trato Gastrointestinal/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Mosquitos Vetores , Glândulas Salivares , Vacinas Atenuadas , Carga Viral , Vírus da Febre Amarela/genética
14.
NPJ Vaccines ; 4: 12, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820355

RESUMO

Following the RTS,S malaria vaccine, which showed only partial protection with short-term memory, there is strong support to develop second-generation malaria vaccines that yield higher efficacy with longer duration. The use of replicating viral vectors to deliver subunit vaccines is of great interest due to their capacity to induce efficient cellular immune responses and long-term memory. The measles vaccine virus offers an efficient and safe live viral vector that could easily be implemented in the field. Here, we produced recombinant measles viruses (rMV) expressing malaria "gold standard" circumsporozoïte antigen (CS) of Plasmodium berghei (Pb) and Plasmodium falciparum (Pf) to test proof of concept of this delivery strategy. Immunization with rMV expressing PbCS or PfCS induced high antibody responses in mice that did not decrease for at least 22 weeks post-prime, as well as rapid development of cellular immune responses. The observed long-term memory response is key for development of second-generation malaria vaccines. Sterile protection was achieved in 33% of immunized mice, as usually observed with the CS antigen, and all other immunized animals were clinically protected from severe and lethal Pb ANKA-induced cerebral malaria. Further rMV-vectored malaria vaccine candidates expressing additional pre-erythrocytic and blood-stage antigens in combination with rMV expressing PfCS may provide a path to development of next generation malaria vaccines with higher efficacy.

15.
Viruses ; 11(3)2019 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-30909570

RESUMO

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related Old-World mammarenaviruses. LASV causes severe hemorrhagic fever with high mortality in humans, whereas no case of MOPV infection has been reported. Comparing MOPV and LASV is a powerful strategy to unravel pathogenic mechanisms that occur during the course of pathogenic arenavirus infection. We used a yeast two-hybrid approach to identify cell partners of MOPV and LASV Z matrix protein in which two autophagy adaptors were identified, NDP52 and TAX1BP1. Autophagy has emerged as an important cellular defense mechanism against viral infections but its role during arenavirus infection has not been shown. Here, we demonstrate that autophagy is transiently induced by MOPV, but not LASV, in infected cells two days after infection. Impairment of the early steps of autophagy significantly decreased the production of MOPV and LASV infectious particles, whereas a blockade of the degradative steps impaired only MOPV infectious particle production. Our study provides insights into the role played by autophagy during MOPV and LASV infection and suggests that this process could partially explain their different pathogenicity.


Assuntos
Arenavirus/fisiologia , Autofagia , Vírus Lassa/fisiologia , Animais , Arenavirus/patogenicidade , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Vírus Lassa/patogenicidade , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Técnicas do Sistema de Duplo-Híbrido , Células Vero
16.
Front Immunol ; 10: 134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809223

RESUMO

The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1-GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.


Assuntos
Antivirais/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Guanabenzo/análogos & derivados , Animais , Antivirais/uso terapêutico , Linhagem Celular , Vírus de DNA/efeitos dos fármacos , Vírus de DNA/fisiologia , Guanabenzo/farmacologia , Guanabenzo/uso terapêutico , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Infecções por Poxviridae/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Vírus de RNA/fisiologia , Coelhos , Infecções Tumorais por Vírus/tratamento farmacológico , Replicação Viral/efeitos dos fármacos
17.
Cancer Immunol Immunother ; 68(4): 533-544, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30656384

RESUMO

Cancer immunotherapy is seeing an increasing focus on vaccination with tumor-associated antigens (TAAs). Human telomerase (hTERT) is a TAA expressed by most tumors to overcome telomere shortening. Tolerance to hTERT can be easily broken both naturally and experimentally and hTERT DNA vaccine candidates have been introduced in clinical trials. DNA prime/boost strategies have been widely developed to immunize efficiently against infectious diseases. We explored the use of a recombinant measles virus (MV) hTERT vector to boost DNA priming as recombinant live attenuated measles virus has an impressive safety and efficacy record. Here, we show that a MV-TERT vector can rapidly and strongly boost DNA hTERT priming in MV susceptible IFNAR/CD46 mouse models. The cellular immune responses were Th1 polarized. No humoral responses were elicited. The 4 kb hTERT transgene did not impact MV replication or induction of cell-mediated responses. These findings validate the MV-TERT vector to boost cell-mediated responses following DNA priming in humans.


Assuntos
Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Vetores Genéticos , Imunidade Celular , Vírus do Sarampo , Linfócitos T/imunologia , Telomerase/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer/genética , Linhagem Celular , Chlorocebus aethiops , Citocinas/metabolismo , Citotoxicidade Imunológica , Vetores Genéticos/genética , Humanos , Imunização , Imunização Secundária , Vírus do Sarampo/genética , Camundongos , Camundongos Transgênicos , Telomerase/genética , Vacinas de DNA , Células Vero
18.
Viruses ; 11(1)2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650657

RESUMO

Flaviviruses, such as dengue (DENV), West Nile (WNV), yellow fever (YFV) and Zika (ZIKV) viruses, are mosquito-borne pathogens that present a major risk to global public health. To identify host factors that promote flavivirus replication, we performed a genome-wide gain-of-function cDNA screen for human genes that enhance the replication of flavivirus reporter particles in human cells. The screen recovered seventeen potential host proteins that promote viral replication, including the previously known dolichyl-diphosphooligosaccharide--protein glycosyltransferase non-catalytic subunit (DDOST). Using silencing approaches, we validated the role of four candidates in YFV and WNV replication: ribosomal protein L19 (RPL19), ribosomal protein S3 (RPS3), DDOST and importin 9 (IPO9). Applying a panel of virological, biochemical and microscopic methods, we validated further the role of RPL19 and DDOST as host factors required for optimal replication of YFV, WNV and ZIKV. The genome-wide gain-of-function screen is thus a valid approach to advance our understanding of flavivirus replication.


Assuntos
Flavivirus/genética , Interações Hospedeiro-Patógeno/genética , Replicação Viral , Animais , Linhagem Celular , Culicidae/virologia , Vírus da Dengue/genética , Flavivirus/fisiologia , Biblioteca Gênica , Hexosiltransferases/genética , Humanos , Carioferinas/genética , Proteínas de Membrana/genética , Proteínas Ribossômicas/genética , Vírus do Nilo Ocidental/genética , Zika virus/genética
19.
Expert Rev Vaccines ; 18(4): 393-403, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30601074

RESUMO

INTRODUCTION: The large global burden of viral infections and especially the rapidly spreading vector-borne diseases and other emerging viral diseases show the need for new approaches in vaccine development. Several new vaccine technology platforms have been developed and are under evaluation. Areas covered: This article discusses the measles vector platform technology derived from the safe and highly efficacious measles virus vaccine. The pipeline of measles-vectored vaccine candidates against viral diseases is reviewed. Particular focus is given to the Chikungunya vaccine candidate as the first measles-vectored vaccine that demonstrated safety, immunogenicity, and functionality of the technology in humans even in the presence of pre-existing anti-measles immunity and thus achieved proof of concept for the technology. Expert commentary: Demonstrating no impact of pre-existing anti-measles immunity in humans on the response to the transgene was fundamental for the technology and indicates that the technology is suitable for large-scale immunization in measles pre-immune populations. The proof of concept in humans combined with a large preclinical track record of safety, immunogenicity, and efficacy for a variety of pathogens suggest the measles vector platform as promising plug-and-play vaccine platform technology for rapid development of effective preventive vaccines against viral and other infectious diseases.


Assuntos
Febre de Chikungunya/prevenção & controle , Vírus Chikungunya/imunologia , Imunização , Vírus do Sarampo/genética , Vacinas Virais/imunologia , Viroses/prevenção & controle , Febre de Chikungunya/virologia , Humanos , Vacinas Sintéticas/imunologia , Vacinas Virais/genética , Viroses/virologia
20.
Viruses ; 12(1)2019 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-31906112

RESUMO

Lassa virus (LASV) and Mopeia virus (MOPV) are two closely related, rodent-born mammarenaviruses. LASV is the causative agent of Lassa fever, a deadly hemorrhagic fever endemic in West Africa, whereas MOPV is non-pathogenic in humans. The Z matrix protein of arenaviruses is essential to virus assembly and budding by recruiting host factors, a mechanism that remains partially defined. To better characterize the interactions involved, a yeast two-hybrid screen was conducted using the Z proteins from LASV and MOPV as a bait. The cellular proteins ITCH and WWP1, two members of the Nedd4 family of HECT E3 ubiquitin ligases, were found to bind the Z proteins of LASV, MOPV and other arenaviruses. The PPxY late-domain motif of the Z proteins is required for the interaction with ITCH, although the E3 ubiquitin-ligase activity of ITCH is not involved in Z ubiquitination. The silencing of ITCH was shown to affect the replication of the old-world mammarenaviruses LASV, MOPV, Lymphocytic choriomeningitis virus (LCMV) and to a lesser extent Lujo virus (LUJV). More precisely, ITCH was involved in the egress of virus-like particles and the release of infectious progeny viruses. Thus, ITCH constitutes a novel interactor of LASV and MOPV Z proteins that is involved in virus assembly and release.


Assuntos
Arenaviridae/fisiologia , Interações entre Hospedeiro e Microrganismos , Vírus Lassa/fisiologia , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/metabolismo , Montagem de Vírus , Animais , Arenaviridae/genética , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Vírus Lassa/genética , Proteínas Repressoras/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Células Vero , Proteínas Virais/genética
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