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1.
J Virol ; 93(19)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31315994

RESUMO

As many tumor cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, frontline cancer therapies often administer anti-VEGF (α-VEGF) antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumor vasculature, we studied the functional tolerance, evasion of neutralization, and induction of α-VEGF antibodies of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the 3-fold capsid spike was replaced by VEGF-blocking peptides: P6L (PQPRPL) and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture, exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti-spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an α-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the 3-fold axis to close up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture into a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow an infectious parvovirus with immune antineovascularization and evasion capacities by replacing an antibody footprint in the capsid 3-fold axis with VEGF-blocking peptides, and it also illustrates the evolutionary capacity of single-stranded DNA (ssDNA) viruses to overcome engineered capsid structural restrictions.IMPORTANCE Targeting the VEGF signaling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumors. VEGF-blocking peptides (VEbp) engineered in the capsid 3-fold axis endowed the infectious parvovirus MVM with the ability to induce α-VEGF antibodies without adjuvant and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restraints that the capsid imposes on the peptide configuration and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp resolved the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. These data show the promise of antineovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses but also raise safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.


Assuntos
Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Vírus Miúdo do Camundongo/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Proteínas do Capsídeo/genética , Camundongos Endogâmicos BALB C , Vírus Miúdo do Camundongo/genética , Vírus Miúdo do Camundongo/crescimento & desenvolvimento , Vírus Oncolíticos/genética , Vírus Oncolíticos/crescimento & desenvolvimento , Vírus Oncolíticos/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Carga Viral , Montagem de Vírus , Ligação Viral , Internalização do Vírus
2.
J Immunol ; 195(9): 4466-4478, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26416276

RESUMO

Dectin-1 (Clec7a) is a paradigmatic C-type lectin receptor that binds Syk through a hemITAM motif and couples sensing of pathogens such as fungi to induction of innate responses. Dectin-1 engagement triggers a plethora of activating events, but little is known about the modulation of such pathways. Trying to define a more precise picture of early Dectin-1 signaling, we explored the interactome of the intracellular tail of the receptor in mouse dendritic cells. We found unexpected binding of SHIP-1 phosphatase to the phosphorylated hemITAM. SHIP-1 colocalized with Dectin-1 during phagocytosis of zymosan in a hemITAM-dependent fashion. Moreover, endogenous SHIP-1 relocated to live or heat-killed Candida albicans-containing phagosomes in a Dectin-1-dependent manner in GM-CSF-derived bone marrow cells (GM-BM). However, SHIP-1 absence in GM-BM did not affect activation of MAPK or production of cytokines and readouts dependent on NF-κB and NFAT. Notably, ROS production was enhanced in SHIP-1-deficient GM-BM treated with heat-killed C. albicans, live C. albicans, or the specific Dectin-1 agonists curdlan or whole glucan particles. This increased oxidative burst was dependent on Dectin-1, Syk, PI3K, phosphoinositide-dependent protein kinase 1, and NADPH oxidase. GM-BM from CD11c∆SHIP-1 mice also showed increased killing activity against live C. albicans that was dependent on Dectin-1, Syk, and NADPH oxidase. These results illustrate the complexity of myeloid C-type lectin receptor signaling, and how an activating hemITAM can also couple to intracellular inositol phosphatases to modulate selected functional responses and tightly regulate processes such as ROS production that could be deleterious to the host.


Assuntos
Motivos de Aminoácidos/imunologia , Candida albicans/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Monoéster Fosfórico Hidrolases/imunologia , Espécies Reativas de Oxigênio/imunologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/microbiologia , Candida albicans/fisiologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Inositol Polifosfato 5-Fosfatases , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos Knockout , Microscopia Confocal , Dados de Sequência Molecular , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Fagossomos/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/imunologia
3.
Science ; 362(6412): 351-356, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30337411

RESUMO

Host injury triggers feedback mechanisms that limit tissue damage. Conventional type 1 dendritic cells (cDC1s) express dendritic cell natural killer lectin group receptor-1 (DNGR-1), encoded by the gene Clec9a, which senses tissue damage and favors cross-presentation of dead-cell material to CD8+ T cells. Here we find that DNGR-1 additionally reduces host-damaging inflammatory responses induced by sterile and infectious tissue injury in mice. DNGR-1 deficiency leads to exacerbated caerulein-induced necrotizing pancreatitis and increased pathology during systemic Candida albicans infection without affecting fungal burden. This effect is B and T cell-independent and attributable to increased neutrophilia in DNGR-1-deficient settings. Mechanistically, DNGR-1 engagement activates SHP-1 and inhibits MIP-2 (encoded by Cxcl2) production by cDC1s during Candida infection. This consequently restrains neutrophil recruitment and promotes disease tolerance. Thus, DNGR-1-mediated sensing of injury by cDC1s serves as a rheostat for the control of tissue damage, innate immunity, and immunopathology.


Assuntos
Candida albicans/imunologia , Candidíase/patologia , Células Dendríticas/imunologia , Lectinas Tipo C/fisiologia , Infiltração de Neutrófilos/imunologia , Pâncreas/patologia , Pancreatite Necrosante Aguda/patologia , Receptores Imunológicos/fisiologia , Animais , Lectinas Tipo C/genética , Camundongos , Camundongos Mutantes , Necrose , Infiltração de Neutrófilos/genética , Pâncreas/imunologia , Pâncreas/microbiologia , Pancreatite Necrosante Aguda/microbiologia , Receptores Imunológicos/genética
4.
J Clin Invest ; 122(5): 1628-43, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22505455

RESUMO

In order to prime T cells, DCs integrate signals emanating directly from pathogens and from their noxious action on the host. DNGR-1 (CLEC9A) is a DC-restricted receptor that detects dead cells. Therefore, we investigated the possibility that DNGR-1 affects immunity to cytopathic viruses. DNGR-1 was essential for cross-presentation of dying vaccinia virus-infected (VACV-infected) cells to CD8(+) T cells in vitro. Following injection of VACV or VACV-infected cells into mice, DNGR-1 detected the ligand in dying infected cells and mediated cross-priming of anti-VACV CD8(+) T cells. Loss of DNGR-1 impaired the CD8+ cytotoxic response to VACV, especially against those virus strains that are most dependent on cross-presentation. The decrease in total anti-VACV CTL activity was associated with a profound increase in viral load and delayed resolution of the primary lesion. In addition, lack of DNGR-1 markedly diminished protection from infection induced by vaccination with the modified vaccinia Ankara (MVA) strain. DNGR-1 thus contributes to anti-VACV immunity, following both primary infection and vaccination. The non-redundant ability of DNGR-1 to regulate cross-presentation of viral antigens suggests that this form of regulation of antiviral immunity could be exploited for vaccination.


Assuntos
Apresentação Cruzada , Células Dendríticas/metabolismo , Lectinas Tipo C/fisiologia , Receptores Imunológicos/fisiologia , Vaccinia virus/imunologia , Vacínia/imunologia , Imunidade Adaptativa , Animais , Apresentação de Antígeno , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Apoptose , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Técnicas de Inativação de Genes , Interferon gama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lisossomos/metabolismo , Lisossomos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Necrose/virologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Quinase Syk , Vacínia/patologia , Vaccinia virus/fisiologia , Carga Viral
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