RESUMO
The blastocyst is the preimplantation stage embryo that consists of two major components: the inner cell mass (ICM) and the trophectoderm (TE). The ICM gives rise to the fetus and some extraembryonic tissues whereas the TE contributes to development of the placenta. Previous studies have demonstrated that both human and mouse embryonic stem cells (ESCs) derived from the ICM are deficient in expressing type I IFNs in response to viral infection. In this study, we investigated the IFN response in mouse trophoblast stem cells (TSCs) and their in vitro differentiated trophoblasts (TSC-TBs). In this study, we report that, unlike ESCs, TSCs have a functional IFN system. They can express type I IFNs in response to viral stimuli and express IFN-stimulated genes in response to type I IFNs. TSC-TBs have a further developed IFN system and acquired the ability to express specialized type III IFN-λ. Furthermore, TSCs and TSC-TBs can provide ESCs with antiviral activity against Chikungunya, West Nile, and Zika virus infection, as demonstrated with a novel coculture model that simulates the temporal and spatial relationship between the ICM and the TE in a blastocyst. Taken together, our data demonstrate that mouse ESCs can respond to type I IFNs and gain IFN-based antiviral protection from TSCs and TSC-TBs via paracrine signaling mechanisms even though they themselves are unable to express type I IFNs.
Assuntos
Infecção por Zika virus , Zika virus , Animais , Antivirais/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias , Feminino , Humanos , Camundongos , Comunicação Parácrina , Gravidez , TrofoblastosRESUMO
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus, and its infection can cause long-term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti-CHIKV monoclonal antibody (mAb) produced in wild-type (WT) and glycoengineered (∆XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ∆XFT carried a single N-glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N-glycans including the typical plant GnGnXF3 glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ∆XFT plant-produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ∆XFT-produced mAbs. This is the first report of the efficacy of plant-produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Febre de Chikungunya/terapia , Nicotiana/metabolismo , Animais , Vírus Chikungunya , Camundongos , Plantas Geneticamente ModificadasRESUMO
Molecular determinants and mechanisms of arthropod-borne flavivirus transmission to the vertebrate host are poorly understood. In this study, we show for the first time that a cell line from medically important arthropods, such as ticks, secretes extracellular vesicles (EVs) including exosomes that mediate transmission of flavivirus RNA and proteins to the human cells. Our study shows that tick-borne Langat virus (LGTV), a model pathogen closely related to tick-borne encephalitis virus (TBEV), profusely uses arthropod exosomes for transmission of viral RNA and proteins to the human- skin keratinocytes and blood endothelial cells. Cryo-electron microscopy showed the presence of purified arthropod/neuronal exosomes with the size range of 30 to 200 nm in diameter. Both positive and negative strands of LGTV RNA and viral envelope-protein were detected inside exosomes derived from arthropod, murine and human cells. Detection of Nonstructural 1 (NS1) protein in arthropod and neuronal exosomes further suggested that exosomes contain viral proteins. Viral RNA and proteins in exosomes derived from tick and mammalian cells were secured, highly infectious and replicative in all tested evaluations. Treatment with GW4869, a selective inhibitor that blocks exosome release affected LGTV loads in both arthropod and mammalian cell-derived exosomes. Transwell-migration assays showed that exosomes derived from infected-brain-microvascular endothelial cells (that constitute the blood-brain barrier) facilitated LGTV RNA and protein transmission, crossing of the barriers and infection of neuronal cells. Neuronal infection showed abundant loads of both tick-borne LGTV and mosquito-borne West Nile virus RNA in exosomes. Our data also suggest that exosome-mediated LGTV viral transmission is clathrin-dependent. Collectively, our results suggest that flaviviruses uses arthropod-derived exosomes as a novel means for viral RNA and protein transmission from the vector, and the vertebrate exosomes for dissemination within the host that may subsequently allow neuroinvasion and neuropathogenesis.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Encefalite Transmitida por Carrapatos/transmissão , Exossomos/virologia , Modelos Biológicos , Neurônios/virologia , RNA Viral/metabolismo , Proteínas Virais/metabolismo , Animais , Vetores Artrópodes/citologia , Vetores Artrópodes/ultraestrutura , Vetores Artrópodes/virologia , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Córtex Cerebral/ultraestrutura , Córtex Cerebral/virologia , Chlorocebus aethiops , Técnicas de Cocultura , Microscopia Crioeletrônica , Embrião de Mamíferos/citologia , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Vírus da Encefalite Transmitidos por Carrapatos/ultraestrutura , Encefalite Transmitida por Carrapatos/patologia , Encefalite Transmitida por Carrapatos/virologia , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Endotélio Vascular/virologia , Exossomos/ultraestrutura , Interações Hospedeiro-Parasita , Interações Hospedeiro-Patógeno , Humanos , Ixodes/citologia , Ixodes/ultraestrutura , Ixodes/virologia , Queratinócitos/citologia , Queratinócitos/patologia , Queratinócitos/ultraestrutura , Queratinócitos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/patologia , Neurônios/ultraestruturaRESUMO
We reported previously that mouse embryonic stem cells do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mouse embryonic stem cells and mouse embryonic stem cell-differentiated cells. We demonstrate that LPS, TNF-α, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NF-κB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mouse embryonic stem cells. Similar results were obtained in human embryonic stem cells. In addition to the inactive state of NF-κB, the deficiency in the inflammatory response in mouse embryonic stem cells is also attributed to the lack of functional receptors for LPS and TNF-α. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NF-κB from its inactive to active state. However, a limited response to TNF-α and viral infection, but not to LPS, was observed in mouse embryonic stem cell-differentiated fibroblasts. We conclude that the inflammatory response mechanism is not active in mouse embryonic stem cells, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this article demonstrate that embryonic stem cells are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology, and embryonic stem cell-based regenerative medicine.
Assuntos
Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Células-Tronco Embrionárias/fisiologia , Inflamação/imunologia , Interferons/metabolismo , NF-kappa B/metabolismo , Viroses/imunologia , Animais , Diferenciação Celular , Imunidade , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos DBA , Células RAW 264.7 , Fator de Necrose Tumoral alfa/imunologiaRESUMO
CD8+ T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8+ T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a-/-) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8+ T cells isolated from Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 postinfection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8+ T cell cytotoxicity, which may have broad implications in other microbial infections and cancers. IMPORTANCE: Interleukin-17A (IL-17A) and CD8+ T cells regulate diverse immune functions in microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while CD8+ T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8+ T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8+ T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces the viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8+ T cell axis may also have broad implications for immunity to other microbial infections and cancers, where CD8+ T cell functions are crucial.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Interleucina-17/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Febre do Nilo Ocidental/tratamento farmacológico , Vírus do Nilo Ocidental/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/imunologia , Encéfalo/virologia , Feminino , Expressão Gênica , Humanos , Interleucina-17/genética , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/virologia , Cultura Primária de Células , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Análise de Sobrevida , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Resultado do Tratamento , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/crescimento & desenvolvimentoRESUMO
West Nile virus (WNV), a mosquito-transmitted single-stranded RNA (ssRNA) flavivirus, causes human disease of variable severity. We investigated Toll-like receptor 7-deficient (Tlr7(-/-)) and myeloid differentiation factor 88-deficient (Myd88(-/-)) mice, which both have defective recognition of ssRNA, and found increased viremia and susceptibility to lethal WNV infection. Despite increased tissue concentrations of most innate cytokines, CD45(+) leukocytes and CD11b(+) macrophages failed to home to WNV-infected cells and infiltrate into target organs of Tlr7(-/-) mice. Tlr7(-/-) mice and macrophages had reduced interleukin-12 (IL-12) and IL-23 responses after WNV infection, and mice deficient in IL-12 p40 and IL-23 p40 (Il12b(-/-)) or IL-23 p19 (Il23a(-/-)), but not IL-12 p35 (Il12a(-/-)), responded similarly to Tlr7(-/-) mice, with increased susceptibility to lethal WNV encephalitis. Collectively, these results demonstrate that TLR7 and IL-23-dependent WNV responses represent a vital host defense mechanism that operates by affecting immune cell homing to infected target cells.
Assuntos
Movimento Celular/imunologia , Glicoproteínas de Membrana/metabolismo , Receptor 7 Toll-Like/metabolismo , Febre do Nilo Ocidental/imunologia , Febre do Nilo Ocidental/metabolismo , Animais , Citocinas/imunologia , Suscetibilidade a Doenças , Interleucina-23/deficiência , Interleucina-23/genética , Interleucina-23/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/imunologia , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genética , Febre do Nilo Ocidental/genética , Febre do Nilo Ocidental/virologiaRESUMO
West Nile virus (WNV) is a neurotropic ssRNA flavivirus that can cause encephalitis, meningitis, and death in humans and mice. Human TLR7 and TLR8 and mouse TLR7 recognize viral ssRNA motifs and induce antiviral immunity. However, the role of mouse TLR8 in antiviral immunity is poorly understood. In this article, we report that TLR8-deficient (Tlr8-/-) mice were resistant to WNV infection compared with wild-type controls. Efficient WNV clearance and moderate susceptibility to WNV-mediated neuronal death in Tlr8-/- mice were attributed to overexpression of Tlr7 and IFN-stimulated gene-56 expression, whereas reduced expression of the proapoptotic gene coding Bcl2-associated X protein was observed. Interestingly, suppressor of cytokine signaling (SOCS)-1 directly associated with TLR8, but not with TLR7, indicating a novel role for TLR8 regulation of SOCS-1 function, whereas selective small interfering RNA knockdown of Socs-1 resulted in induced IFN-stimulated gene-56 and Tlr7 expression following WNV infection. Collectively, we report that TLR8 coupling with SOCS-1 inhibits TLR7-mediated antiviral immunity during WNV infection in mice.
Assuntos
Proteína 1 Supressora da Sinalização de Citocina/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Camundongos , Camundongos Knockout , Proteína 1 Supressora da Sinalização de Citocina/genética , Receptor 7 Toll-Like/genética , Receptor 8 Toll-Like/genética , Febre do Nilo Ocidental/genéticaRESUMO
The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.
Assuntos
Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Facilitadores/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Nicotiana/genética , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Dengue/virologia , Vírus da Dengue/genética , Expressão Gênica , Humanos , Nicotiana/metabolismoRESUMO
Embryonic stem cells (ESCs) represent a promising cell source for regenerative medicine. Intensive research over the past 2 decades has led to the feasibility of using ESC-differentiated cells (ESC-DCs) in regenerative medicine. However, increasing evidence indicates that ESC-DCs generated by current differentiation methods may not have equivalent cellular functions to their in vivo counterparts. Recent studies have revealed that both human and mouse ESCs as well as some types of ESC-DCs lack or have attenuated innate immune responses to a wide range of infectious agents. These findings raise important concerns for their therapeutic applications since ESC-DCs, when implanted to a wound site of a patient, where they would likely be exposed to pathogens and inflammatory cytokines. Understanding whether an attenuated immune response is beneficial or harmful to the interaction between host and grafted cells becomes an important issue for ESC-based therapy. A substantial amount of recent evidence has demonstrated that the lack of innate antiviral responses is a common feature to ESCs and other types of pluripotent cells. This has led to the hypothesis that mammals may have adapted different antiviral mechanisms at different stages of organismal development. The underdeveloped innate immunity represents a unique and uncharacterized property of ESCs that may have important implications in developmental biology, immunology, and in regenerative medicine.
Assuntos
Biologia do Desenvolvimento/tendências , Células-Tronco Embrionárias/imunologia , Imunidade Inata/imunologia , Medicina Regenerativa/tendências , Animais , Diferenciação Celular/imunologia , Humanos , Células-Tronco Pluripotentes/imunologiaRESUMO
We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFNs) in response to viral infection and synthetic viral RNA analogs (Wang, R., Wang, J., Paul, A. M., Acharya, D., Bai, F., Huang, F., and Guo, Y. L. (2013) J. Biol. Chem. 288, 15926-15936). Here, we report that mESCs are able to respond to type I IFNs, express IFN-stimulated genes, and mediate the antiviral effect of type I IFNs against La Crosse virus and chikungunya virus. The major signaling components in the IFN pathway are expressed in mESCs. Therefore, the basic molecular mechanisms that mediate the effects of type I IFNs are functional in mESCs; however, these mechanisms may not yet be fully developed as mESCs express lower levels of IFN-stimulated genes and display weaker antiviral activity in response to type I IFNs when compared with fibroblasts. Further analysis demonstrated that type I IFNs do not affect the stem cell state of mESCs. We conclude that mESCs are deficient in type I IFN expression, but they can respond to and mediate the cellular effects of type I IFNs. These findings represent unique and uncharacterized properties of mESCs and are important for understanding innate immunity development and ESC physiology.
Assuntos
Antivirais/imunologia , Células-Tronco Embrionárias/imunologia , Fibroblastos/imunologia , Interferon Tipo I/imunologia , Animais , Antivirais/metabolismo , Antivirais/farmacologia , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Vírus Chikungunya/imunologia , Vírus Chikungunya/fisiologia , Chlorocebus aethiops , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/virologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferon Tipo I/metabolismo , Interferon Tipo I/farmacologia , Vírus La Crosse/imunologia , Vírus La Crosse/fisiologia , Camundongos , Microscopia Confocal , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitinas/genética , Ubiquitinas/imunologia , Ubiquitinas/metabolismo , Células VeroRESUMO
Embryonic stem cells (ESCs) are considered to be a promising cell source for regenerative medicine because of their unlimited capacity for self-renewal and differentiation. However, little is known about the innate immunity in ESCs and ESC-derived cells. We investigated the responses of mouse (m)ESCs to three types of live viruses as follows: La Crosse virus, West Nile virus, and Sendai virus. Our results demonstrated mESCs were susceptible to viral infection, but they were unable to express type I interferons (IFNα and IFNß, IFNα/ß), which differ from fibroblasts (10T1/2 cells) that robustly express IFNα/ß upon viral infections. The failure of mESCs to express IFNα/ß was further demonstrated by treatment with polyIC, a synthetic viral dsRNA analog that strongly induced IFNα/ß in 10T1/2 cells. Although polyIC transiently inhibited the transcription of pluripotency markers, the stem cell morphology was not significantly affected. However, polyIC can induce dsRNA-activated protein kinase in mESCs, and this activation resulted in a strong inhibition of cell proliferation. We conclude that the cytosolic receptor dsRNA-activated protein kinase is functional, but the mechanisms that mediate type I IFN expression are deficient in mESCs. This conclusion is further supported by the findings that the major viral RNA receptors are either expressed at very low levels (TLR3 and MDA5) or may not be active (retinoic acid-inducible gene I) in mESCs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Interferon-alfa/biossíntese , Interferon beta/biossíntese , Células-Tronco Pluripotentes/metabolismo , Infecções por Vírus de RNA/metabolismo , Vírus de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/imunologia , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/imunologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/imunologia , Células-Tronco Embrionárias/virologia , Humanos , Indutores de Interferon/farmacologia , Helicase IFIH1 Induzida por Interferon , Interferon-alfa/imunologia , Interferon beta/imunologia , Camundongos , Células-Tronco Pluripotentes/imunologia , Células-Tronco Pluripotentes/virologia , Poli I-C/farmacologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/farmacologia , RNA Viral/imunologia , RNA Viral/farmacologia , Receptor 3 Toll-Like/biossíntese , Receptor 3 Toll-Like/imunologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP-siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP-siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy.
Assuntos
Antivirais/metabolismo , Vírus da Dengue/fisiologia , Portadores de Fármacos/metabolismo , Nanopartículas/metabolismo , RNA Interferente Pequeno/metabolismo , Liberação de Vírus , Replicação Viral , Animais , Chlorocebus aethiops , Vírus da Dengue/genética , Portadores de Fármacos/química , Ouro/metabolismo , Nanopartículas/química , RNA Interferente Pequeno/genética , Células VeroRESUMO
Influenza seasons occur annually, building immune history for individuals, but the influence of this history on subsequent influenza vaccine protection remains unclear. We extracted data from an animal trial to study its potential impact. The trial involved 80 ferrets, each receiving either one type of infection or a placebo before vaccination. We quantified the vaccine protection by evaluating hemagglutination inhibition (HAI) antibody titer responses. We tested whether hosts with different infection histories exhibited similar level of responses when receiving the same vaccine for all homologous and heterologous outcomes. We observed that different pre-existing immunities were generally beneficial to vaccine induced responses, but varied in magnitude. Without pre-immunity, post-vaccination HAI titers after the 1st dose of the vaccine were less likely to be above 1:40, and a booster shot was needed. Our study suggests that pre-existing immunity may strengthen and extend the homologous and heterologous vaccine responses.
Assuntos
Anticorpos Antivirais , Furões , Testes de Inibição da Hemaglutinação , Vacinas contra Influenza , Infecções por Orthomyxoviridae , Animais , Furões/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Formação de Anticorpos/imunologia , Vacinação , Masculino , FemininoRESUMO
To develop an inhalable drug delivery system, we synthesized poly (lactic-co-glycolic acid) nanoparticles with Remdesivir (RDV NPs) as an antiviral agent against SARS-CoV-2 replication and formulated Remdesivir-loaded nanocomposites (RDV NCs) via coating of RDV NPs with novel supramolecular cell-penetrating peptide nanofibers (NFs) to enhance cellular uptake and intracellular drug delivery. RDV NPs and RDV NCs were characterized using variou techniques, including Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and fluorescent microscopy. The cytotoxicity of RDV NCs was assessed in Vero E6 cells and primary human lung epithelial cells, with no significant cytotoxicity observed up to 1000 µg mL-1 and 48 h. RDV NCs were spherically shaped with a size range of 200-300 nm and a zeta potential of â¼+31 mV as well as indicating the presence of coated nanofibers. Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), immunofluorescence and plaque assays of SARS-CoV-2 infected Vero E6 treated with RDV NCs showed significantly higher antiviral activities compared to those of free drug and uncoated RDV NPs. RDV NCs exhibited high antiviral activity against SARS-CoV-2, and the nanocomposite platform has the potential to be developed into an inhalable drug delivery system for other viral infections in the lungs.
RESUMO
Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Compostos Azo , Fibroblastos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos , Azul Tripano/química , Azul Tripano/farmacologia , Regulação Alostérica/efeitos dos fármacos , Antígenos de Diferenciação de Linfócitos B/química , Compostos Azo/química , Compostos Azo/farmacologia , Células Cultivadas , Fibroblastos/citologia , Antígenos de Histocompatibilidade Classe II/química , Humanos , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de ProteínaRESUMO
The plaque-forming assay is a gold standard technique to determine the concentration of infectious viral particles. In this assay, lytic viruses infect and lyse the cells but are immobilized due to the presence of an agarose-containing overlay medium. The progeny viruses can only spread locally to and kill the adjacent cells and finally form a clear zone or plaque after staining the live cells. The number of plaques formed can be theoretically considered as the initial number of the infectious viral particles present in the sample and hence can be expressed as plaque-forming units (PFU) in a volume of the sample. Here, we provide a step-by-step method to carry out a plaque-forming assay to determine the titer of West Nile virus in a cell culture medium, which also can be adapted to other lytic viruses of eukaryotic cells.
Assuntos
Vírus do Nilo Ocidental , Ensaio de Placa ViralRESUMO
Primary neurons are very valuable cells to study the pathogenesis of neurotropic viruses, such as West Nile virus. The mouse primary neurons can be used to assess viral infection profiles and cellular immune responses to a viral infection. However, successful isolation and culture of mouse neurons can be challenging. Here, we report a step-by-step method to prepare a primary neuron culture from adult mice.
Assuntos
Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Camundongos , Neurônios/fisiologia , Imunidade CelularRESUMO
West Nile virus (WNV) is a mosquito-borne, single-stranded, positive-sense RNA virus belonging to the Flaviviridae family. After WNV gains entry through an infected mosquito bite, it replicates in a variety of human cell types and produces a viremia. Although the majority of infected individuals remain asymptomatic, the manifested symptoms in some people range from a mild fever to severe neurological disorder with high morbidity and mortality. In addition, many who recover from WNV neuroinvasive infection present with long-term deficits, including weakness, fatigue, and cognitive problems. Since entering the USA in 1999, WNV has become the most common mosquito-borne virus in North America. Despite the intensive research over 20 years, there are still no approved vaccines or specific treatments for humans, and it remains an urgent need to understand the pathogenesis of WNV and develop specific therapeutics and vaccines.
Assuntos
Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Humanos , Vírus do Nilo Ocidental/genética , Febre do Nilo Ocidental/diagnóstico , Viremia , América do NorteRESUMO
West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV.
RESUMO
Monoclonal antibodies (mAb) against the envelope (E) protein of Zika virus (ZIKV) have shown great potential as therapeutics against the Zika epidemics. However, their use as a therapy may predispose treated individuals to severe infection by the related dengue virus (DENV) via antibody-dependent enhancement of infection (ADE). Here, we generated a broadly neutralizing flavivirus mAb, ZV1, with an identical protein backbone but different Fc glycosylation profiles. The three glycovariants, produced in wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants and in Chinese hamster ovary cells (ZV1WT, ZV1ΔXF, and ZV1CHO), respectively, showed equivalent neutralization potency against both ZIKV and DENV. By contrast, the three mAb glycoforms demonstrated drastically different ADE activity for DENV and ZIKV infection. While ZV1CHO and ZV1ΔXF showed ADE activity upon DENV and ZIKV infection, ZV1WT totally forwent its ADE. Importantly, all three glycovariants exhibited antibody-dependent cellular cytotoxicity (ADCC) against virus-infected cells, with increased potency by the fucose-free ZV1ΔXF glycoform. Moreover, the in vivo efficacy of the ADE-free ZV1WT was demonstrated in a murine model. Collectively, we demonstrated the feasibility of modulating ADE by Fc glycosylation, thereby establishing a novel approach for improving the safety of flavivirus therapeutics. Our study also underscores the versatile use of plants for the rapid expression of complex human proteins to reveal novel insight into antibody function and viral pathogenesis.