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1.
Adv Virus Res ; 104: 283-312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31439151

RESUMO

In this chapter, we present an overview on betaherpesvirus entry, with a focus on human cytomegalovirus, human herpesvirus 6A and human herpesvirus 6B. Human cytomegalovirus (HCMV) is a complex human pathogen with a genome of 235kb encoding more than 200 genes. It infects a broad range of cell types by switching its viral ligand on the virion, using the trimer gH/gL/gO for infection of fibroblasts and the pentamer gH/gL/UL128/UL130/UL131 for infection of other cells such as epithelial and endothelial cells, leading to membrane fusion mediated by the fusion protein gB. Adding to this scenario, however, accumulating data reveal the actual complexity in the viral entry process of HCMV with an intricate interplay among viral and host factors. Key novel findings include the identification of entry receptors platelet-derived growth factor-α receptor (PDGFRα) and Netropilin-2 (Nrp2) for trimer and pentamer, respectively, the determination of atomic structures of the fusion protein gB and the pentamer, and the in situ visualization of the state and arrangement of functional glycoproteins on virion. This is covered in the first part of this review. The second part focusses on HHV-6 which is a T lymphotropic virus categorized as two distinct virus species, HHV-6A and HHV-6B based on differences in epidemiological, biological, and immunological aspects, although homology of their entire genome sequences is nearly 90%. HHV-6B is a causative agent of exanthema subitum (ES), but the role of HHV-6A is unknown. HHV-6B reactivation occasionally causes encephalitis in patients with hematopoietic stem cell transplant. The HHV-6 specific envelope glycoprotein complex, gH/gL/gQ1/gQ2 is a viral ligand for the entry receptor. Recently, each virus has been found to recognize a different cellular receptor, CD46 for HHV 6A amd CD134 for HHV 6B. These findings show that distinct receptor recognition differing between both viruses could explain their different pathogenesis.


Assuntos
Citomegalovirus/fisiologia , Herpesvirus Humano 6/fisiologia , Internalização do Vírus , Células Endoteliais/virologia , Células Epiteliais/virologia , Fibroblastos/virologia , Glicoproteínas/metabolismo , Humanos , Proteína Cofatora de Membrana/metabolismo , Neuropilina-2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores OX40/metabolismo , Receptores Virais/metabolismo , Linfócitos T/virologia , Proteínas do Envelope Viral/metabolismo
2.
Int J Oncol ; 55(2): 347-358, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31268165

RESUMO

Virotherapy comprises a novel therapeutic approach to selectively eliminate cancer cells. Preclinical, as well as clinical data have demonstrated the efficacy of tumor­selective (oncolytic) viruses in hematological malignancies. In this study, we infected AML cell lines and primary AML cells from patients with measles vaccine virus either expressing GFP or armed with super cytosine deaminase, which converts the prodrug, 5­fluorocytosine, into the chemotherapeutic compound, 5­fluorouracil. Target cell density of the measles entry receptor, CD46, infection rates of targeted leukemic cells, tumor cell viability, and apoptotic rates were determined. We found that measles vaccine virus infected the leukemic blasts and profoundly diminished the number and viability of leukemic cells via the induction of apoptosis. The conversion of 5­fluorocytosine to 5­fluorouracil exerted a potent additive tumoricidal effect. This was also observed in cases when leukemic cells displayed only moderate susceptibility to the oncolytic virus and hence direct oncolysis. Taken together, in this study, we provide a first characterization of the combinatorial use of measles vaccine virus and 5­fluorouracil for treatment of AML. Our approach to site­specifically produce the active drug and combine this agent with the direct lytic effect of virotherapy may overcome present limitations and constitutes a feasible method with which to introduce 5­fluorouracil in the treatment of AML.


Assuntos
Fluoruracila/administração & dosagem , Leucemia Mieloide Aguda/terapia , Vírus do Sarampo/genética , Proteína Cofatora de Membrana/genética , Terapia Viral Oncolítica , Pró-Fármacos/administração & dosagem , Adulto , Idoso , Terapia Combinada , Feminino , Flucitosina/metabolismo , Fluoruracila/metabolismo , Seguimentos , Genes Transgênicos Suicidas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Proteína Cofatora de Membrana/metabolismo , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
4.
Comput Biol Chem ; 80: 384-389, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31112822

RESUMO

The hemagglutinin (H) protein of measles viruses (MeV) mediates binding to the cellular receptors, CD46,human signaling lymphocyte activation molecule and nectin-4. Vaccine strains primarily contain H-proteins possessing MeV-H: Y481 and can utilize CD46. Reports suggest that a single amino acid change in MeV-H at position 481 in wild type strains renders them inefficient in utilizing CD46. The in-depth molecular mechanism by which substitutions at 481 and another reported critical residue position 546 affects CD46 binding affinity however remains elusive. We used molecular docking studies of CD46 with MeV-H possessing Y481 N/D to understand the in-depth molecular mechanism involved. It was found that loss in either of the hydrogen bond (H-bond) contacts (MeV-H:481-CD46:65, MeV-H:546-CD46:63) in the central contact region prevented efficient CD46 binding. Y481 N could form the specific H-bond, while G546S H-bond could be formed only in conjunction with Y481, revealing the significance of these residues in determining CD46 receptor binding potential. Elucidating the underlying molecular mechanism of receptor usage by the MeV has implications to understanding cellular tropism, viral pathogenesis and therapy.


Assuntos
Hemaglutininas Virais/metabolismo , Vírus do Sarampo/química , Proteína Cofatora de Membrana/metabolismo , Receptores Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Hemaglutininas Virais/química , Humanos , Ligação de Hidrogênio , Proteína Cofatora de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores Virais/química
5.
Nat Commun ; 10(1): 741, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30765704

RESUMO

Adenovirus based vectors are of increasing importance for wide ranging therapeutic applications. As vaccines, vectors derived from human adenovirus species D serotypes 26 and 48 (HAdV-D26/48) are demonstrating promising efficacy as protective platforms against infectious diseases. Significant clinical progress has been made, yet definitive studies underpinning mechanisms of entry, infection, and receptor usage are currently lacking. Here, we perform structural and biological analysis of the receptor binding fiber-knob protein of HAdV-D26/48, reporting crystal structures, and modelling putative interactions with two previously suggested attachment receptors, CD46 and Coxsackie and Adenovirus Receptor (CAR). We provide evidence of a low affinity interaction with CAR, with modelling suggesting affinity is attenuated through extended, semi-flexible loop structures, providing steric hindrance. Conversely, in silico and in vitro experiments are unable to provide evidence of interaction between HAdV-D26/48 fiber-knob with CD46, or with Desmoglein 2. Our findings provide insight into the cell-virus interactions of HAdV-D26/48, with important implications for the design and engineering of optimised Ad-based therapeutics.


Assuntos
Infecções por Adenoviridae/metabolismo , Adenovírus Humanos/metabolismo , Proteínas do Capsídeo/metabolismo , Receptores Virais/metabolismo , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/classificação , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/química , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Cristalografia por Raios X , Variação Genética , Humanos , Proteína Cofatora de Membrana/química , Proteína Cofatora de Membrana/metabolismo , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Receptores Virais/química , Homologia de Sequência de Aminoácidos
6.
J Virol ; 93(1)2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30333171

RESUMO

Human adenoviruses (HAdVs) are being explored as vectors for gene transfer and vaccination. Human adenovirus type 26 (HAdV26), which belongs to the largest subgroup of adenoviruses, species D, has a short fiber and a so-far-unknown natural tropism. Due to its low seroprevalence, HAdV26 has been considered a promising vector for the development of vaccines. Despite the fact that the in vivo safety and immunogenicity of HAdV26 have been extensively studied, the basic biology of the virus with regard to receptor use, cell attachment, internalization, and intracellular trafficking is poorly understood. In this work, we investigated the roles of the coxsackievirus and adenovirus receptor (CAR), CD46, and αv integrins in HAdV26 infection of human epithelial cell lines. By performing different gain- and loss-of-function studies, we found that αvß3 integrin is required for efficient infection of epithelial cells by HAdV26, while CAR and CD46 did not increase the transduction efficiency of HAdV26. By studying intracellular trafficking of fluorescently labeled HAdV26 in A549 cells and A549-derived cell clones with stably increased expression of αvß3 integrin, we observed that HAdV26 colocalizes with αvß3 integrin and that increased αvß3 integrin enhances internalization of HAdV26. Thus, we conclude that HAdV26 uses αvß3 integrin as a receptor for infecting epithelial cells. These results give us new insight into the HAdV26 infection pathway and will be helpful in further defining HAdV-based vector manufacturing and vaccination strategies.IMPORTANCE Adenovirus-based vectors are used today for gene transfer and vaccination. HAdV26 has emerged as a promising candidate vector for development of vaccines due to its relatively low seroprevalence and its ability to induce potent immune responses against inserted transgenes. However, data regarding the basic biology of the virus, like receptor usage or intracellular trafficking, are limited. In this work, we found that efficient infection of human epithelial cell lines by HAdV26 requires the expression of the αvß3 integrin. By studying intracellular trafficking of fluorescently labeled HAdV26 in a cell clone with stably increased expression of αvß3 integrin, we observed that HAdV26 colocalizes with αvß3 integrin and confirmed that αvß3 integrin expression facilitates efficient HAdV26 internalization. These results will allow further improvement of HAdV26-based vectors for gene transfer and vaccination.


Assuntos
Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/patogenicidade , Células Epiteliais/metabolismo , Integrina alfaVbeta3/metabolismo , Células A549 , Infecções por Adenovirus Humanos/metabolismo , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Células Epiteliais/citologia , Células Epiteliais/virologia , Humanos , Proteína Cofatora de Membrana/metabolismo , Internalização do Vírus
7.
Mol Pharm ; 16(1): 292-304, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30452273

RESUMO

Brain metastasis is a frequent complication of cancer and may be mediated, at least in part, by the internalization of cancer-cell-derived exosomes into brain capillary endothelial cells. Clarifying the mechanism(s) of this internalization is of interest because it could help us to develop ways to block brain metastasis, as well as affording a potential new route for drug delivery into the brain. Therefore, the purpose of the present study was to address this issue by identifying the receptors involved in the internalization of exosomes derived from a brain-metastatic cancer cell line (SK-Mel-28) into human blood-brain barrier endothelial cells (hCMEC/D3 cells). The combination of sulfo-SBED-based cross-linking and comprehensive proteomics yielded 20 proteins as exosome receptor candidates in hCMEC/D3 cells. The uptake of PKH67-labeled exosomes by hCMEC/D3 cells measured at 37 °C was significantly reduced by 95.6% at 4 °C and by 15.3% in the presence of 1 mM RGD peptide, an integrin ligand. Therefore, we focused on the identified RGD receptors, integrin α5 and integrin αV, and CD46, which is reported to act as an adenovirus receptor, together with integrin αV. A mixture of neutralizing antibodies against integrin α5 and integrin αV significantly decreased the exosome uptake by 11.8%, while application of CD46 siRNA reduced it by 39.0%. Immunohistochemical analysis confirmed the presence of CD46 in human brain capillary endothelial cells. These results suggest that CD46 is a major receptor for the uptake of SK-Mel-28-derived exosomes by human blood-brain barrier endothelial cells (hCMEC/D3 cells).


Assuntos
Encéfalo/metabolismo , Exossomos/metabolismo , Proteômica/métodos , Transporte Biológico/fisiologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Humanos , Melanoma/metabolismo , Proteína Cofatora de Membrana/metabolismo , RNA Interferente Pequeno , Receptores Virais/metabolismo
8.
Nature ; 564(7736): 430-433, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30518863

RESUMO

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation7.


Assuntos
Transplante de Coração , Xenoenxertos/transplante , Papio , Suínos , Transplante Heterólogo , Animais , Anticorpos/análise , Anticorpos/sangue , Proteínas do Sistema Complemento/análise , Enzimas/sangue , Fibrina/análise , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Xenoenxertos/patologia , Humanos , Fígado/enzimologia , Masculino , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/metabolismo , Miocárdio/enzimologia , Necrose , Perfusão , Contagem de Plaquetas , Tempo de Protrombina , Trombomodulina/genética , Trombomodulina/metabolismo , Fatores de Tempo
9.
Blood Adv ; 2(23): 3492-3505, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30530776

RESUMO

In this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53 abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53 wt cell lines (P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53 wt cell lines, but not the TP53 abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency.


Assuntos
Vírus do Sarampo/fisiologia , Proteína Cofatora de Membrana/metabolismo , Mieloma Múltiplo/patologia , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Proteína Cofatora de Membrana/antagonistas & inibidores , Proteína Cofatora de Membrana/genética , MicroRNAs/metabolismo , Mieloma Múltiplo/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/química , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética
10.
Front Immunol ; 9: 2803, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30574140

RESUMO

The aberrant expression of human endogenous retrovirus (HERV) elements of the HERV-W family has been associated with different diseases, including multiple sclerosis (MS). In particular, the expression of the envelope protein (Env) from the multiple sclerosis-associated retrovirus (MSRV), a member of HERV-W family and known for its potent proinflammatory activity, is repeatedly detected in the brain lesions and blood of MS patients. Furthermore, human herpesvirus 6 (HHV-6) infection has long been suspected to play a role in the pathogenesis of MS and neuroinflammation. We show here that both HHV-6A and stimulation of its receptor, transmembrane glycoprotein CD46, induce the expression of MSRV-Env. The engagement of extracellular domains SCR3 and SCR4 of CD46-Cyt1 isoform was required for MSRV-env transactivation, limiting thus the MSRV-Env induction to the CD46 ligands binding these domains, including C3b component of complement, specific monoclonal antibodies, and both infectious and UV-inactivated HHV-6A, but neither HHV-6B nor measles virus vaccine strain. Induction of MSRV-Env required CD46 Cyt-1 singling and was abolished by the inhibitors of protein kinase C. Finally, both membrane-expressed and secreted MSRV-Env trigger TLR4 signaling, displaying thus a proinflammatory potential, characteristic for this viral protein. These data expand the specter of HHV-6A effects in the modulation of the immune response and support the hypothesis that cross-talks between exogenous and endogenous viruses may contribute to inflammatory diseases and participate in neuroinflammation. Furthermore, they reveal a new function of CD46, known as an inhibitor of complement activation and receptor for several pathogens, in transactivation of HERV env genes, which may play an important role in the pathogenesis of inflammatory diseases.


Assuntos
Retrovirus Endógenos , Herpesvirus Humano 6 , Proteína Cofatora de Membrana , Esclerose Múltipla , Proteínas da Gravidez , Infecções por Roseolovirus , Linhagem Celular Tumoral , Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Retrovirus Endógenos/metabolismo , Herpesvirus Humano 6/imunologia , Herpesvirus Humano 6/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Proteína Cofatora de Membrana/imunologia , Proteína Cofatora de Membrana/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/virologia , Proteínas da Gravidez/biossíntese , Proteínas da Gravidez/genética , Proteínas da Gravidez/imunologia , Domínios Proteicos , Infecções por Roseolovirus/genética , Infecções por Roseolovirus/imunologia , Infecções por Roseolovirus/metabolismo
11.
Front Immunol ; 9: 2449, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405635

RESUMO

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity-but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn -/- mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an "upstream" activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.


Assuntos
Catepsina L/metabolismo , Complemento C3a/imunologia , Complemento C3b/imunologia , Cisteína Endopeptidases/metabolismo , Interferon gama/metabolismo , Células Th1/imunologia , Animais , Proliferação de Células , Cisteína Endopeptidases/genética , Humanos , Interferon gama/biossíntese , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteína Cofatora de Membrana/metabolismo , Camundongos , Camundongos Knockout , Receptores de Complemento/metabolismo , Timalfasina/metabolismo
12.
Nat Commun ; 9(1): 4186, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30305631

RESUMO

The induction of human CD4+ Th1 cells requires autocrine stimulation of the complement receptor CD46 in direct crosstalk with a CD4+ T cell-intrinsic NLRP3 inflammasome. However, it is unclear whether human cytotoxic CD8+ T cell (CTL) responses also rely on an intrinsic complement-inflammasome axis. Here we show, using CTLs from patients with CD46 deficiency or with constitutively-active NLRP3, that CD46 delivers co-stimulatory signals for optimal CTL activity by augmenting nutrient-influx and fatty acid synthesis. Surprisingly, although CTLs express NLRP3, a canonical NLRP3 inflammasome is not required for normal human CTL activity, as CTLs from patients with hyperactive NLRP3 activity function normally. These findings establish autocrine complement and CD46 activity as integral components of normal human CTL biology, and, since CD46 is only present in humans, emphasize the divergent roles of innate immune sensors between mice and men.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Ácidos Graxos/metabolismo , Proteína Cofatora de Membrana/metabolismo , Receptores de Complemento/metabolismo , Comunicação Autócrina , Linfócitos T CD4-Positivos/imunologia , Síndromes Periódicas Associadas à Criopirina/imunologia , Síndromes Periódicas Associadas à Criopirina/patologia , Humanos , Ativação Linfocitária/imunologia , Modelos Biológicos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia
13.
Sci Rep ; 8(1): 13442, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30194327

RESUMO

More than 70 human adenoviruses with type-dependent pathogenicity have been identified but biological information about the majority of these virus types is scarce. Here we employed multiple sequence alignments and structural information to predict receptor usage for the development of an adenoviral vector with novel biological features. We report the generation of a cloned adenovirus based on human adenovirus type 17 (HAdV17) with high sequence homology to the well characterized human adenovirus type 37 (HAdV37) that causes epidemic keratoconjunctivitis (EKC). Our study revealed that human CD46 (CD46) is involved in cell entry of HAdV17. Moreover, we found that HAdV17 infects endothelial cells (EC) in vitro including primary cells at higher efficiencies compared to the commonly used human adenovirus type 5 (HAdV5). Using a human CD46 transgenic mouse model, we observed that HAdV17 displays a broad tropism in vivo after systemic injection and that it transduces ECs in this mouse model. We conclude that the HAdV17-based vector may provide a novel platform for gene therapy.


Assuntos
Adenovírus Humanos/fisiologia , Células Endoteliais , Proteína Cofatora de Membrana/metabolismo , Transdução Genética , Tropismo Viral/fisiologia , Internalização do Vírus , Animais , Células CHO , Cricetulus , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/virologia , Vetores Genéticos , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Jurkat , Proteína Cofatora de Membrana/genética , Camundongos Transgênicos
14.
JCI Insight ; 3(17)2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30185663

RESUMO

Although initially responsive to androgen signaling inhibitors (ASIs), metastatic castration-resistant prostate cancer (mCRPC) inevitably develops and is incurable. In addition to adenocarcinoma (adeno), neuroendocrine prostate cancer (NEPC) emerges to confer ASI resistance. We have previously combined laser capture microdissection and phage antibody display library selection on human cancer specimens and identified novel internalizing antibodies binding to tumor cells residing in their tissue microenvironment. We identified the target antigen for one of these antibodies as CD46, a multifunctional protein that is best known for negatively regulating the innate immune system. CD46 is overexpressed in primary tumor tissue and CRPC (localized and metastatic; adeno and NEPC), but expressed at low levels on normal tissues except for placental trophoblasts and prostate epithelium. Abiraterone- and enzalutamide-treated mCRPC cells upregulate cell surface CD46 expression. Genomic analysis showed that the CD46 gene is gained in 45% abiraterone-resistant mCRPC patients. We conjugated a tubulin inhibitor to our macropinocytosing anti-CD46 antibody and showed that the resulting antibody-drug conjugate (ADC) potently and selectively kills both adeno and NEPC cell lines in vitro (sub-nM EC50) but not normal cells. CD46 ADC regressed and eliminated an mCRPC cell line xenograft in vivo in both subcutaneous and intrafemoral models. Exploratory toxicology studies of the CD46 ADC in non-human primates demonstrated an acceptable safety profile. Thus, CD46 is an excellent target for antibody-based therapy development, which has potential to be applicable to both adenocarcinoma and neuroendocrine types of mCRPC that are resistant to current treatment.


Assuntos
Adenocarcinoma/metabolismo , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/metabolismo , Proteína Cofatora de Membrana/metabolismo , Tumores Neuroendócrinos/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Androstenos/farmacologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Afinidade de Anticorpos , Antígenos de Neoplasias/imunologia , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Macaca fascicularis , Masculino , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/imunologia , Tumores Neuroendócrinos/tratamento farmacológico , Tumores Neuroendócrinos/imunologia , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Proteínas Recombinantes de Fusão , Transdução de Sinais/efeitos dos fármacos , Terapêutica , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Int J Mol Sci ; 19(9)2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-30201920

RESUMO

CD46 is generally overexpressed in many human cancers, representing a prime target for CD46-binding adenoviruses (Ads). This could help to overcome low anti-tumoral activity by coxsackie-adenoviral receptor (CAR)-targeting cancer gene therapy viruses. However, because of scarce side-by-side information about CAR and CD46 expression levels in cancer cells, mixed observations of cancer therapeutic efficacy have been observed. This study evaluated Ad-mediated therapeutic efficacy using either CAR-targeting Ad5 or CD46-targeting Ad5/35 fiber chimera in bladder cancer cell lines. Compared with normal urothelia, bladder cancer tissue generally overexpressed both CAR and CD46. While CAR expression was not correlated with disease progression, CD46 expression was inversely correlated with tumor grade, stage, and risk grade. In bladder cancer cell lines, expression levels of CD46 and CAR were highly correlated with Ad5/35- and Ad5-mediated gene transduction and cytotoxicity, respectively. In a human EJ bladder cancer xenograft mouse model, with either overexpressed or suppressed CD46 expression levels, Ad5/35-tk followed by ganciclovir (GCV) treatment significantly affected tumor growth, whereas Ad5-tk/GCV had only minimal effects. Overall, our findings suggest that bladder cancer cells overexpress both CAR and CD46, and that adenoviral cancer gene therapy targeting CD46 represents a more suitable therapy option than a CAR-targeting therapy, especially in patients with low risk bladder cancers.


Assuntos
Adenoviridae/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Proteína Cofatora de Membrana/metabolismo , Timidina Quinase/genética , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/terapia , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Ganciclovir/administração & dosagem , Ganciclovir/farmacologia , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Vetores Genéticos/administração & dosagem , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Análise de Sobrevida , Transdução Genética , Regulação para Cima , Neoplasias da Bexiga Urinária/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Virology ; 524: 151-159, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30199752

RESUMO

Measles virus has been successfully attenuated on chicken embryo cells to obtain a highly efficient and safe live attenuated vaccine, administered thus far to billions of children. Measles virus attenuation has long been described to involve a modification of cellular tropism with the use of human CD46 ubiquitous receptor. Nevertheless, the use of this receptor in vivo is not obvious. In this study we use four different mouse models to decipher the respective part of hCD46 receptor and type-I interferon response in measles host restriction. We observed that only type-I interferon restricts viral replication of attenuated MV Schwarz strain in mice, independently of the presence of hCD46 receptor. By comparing measles virus immunogenicity in the different models, we confirmed that there was no impact on the absence of this receptor on the immune response. Therefore, we propose to simplify the mouse model.


Assuntos
Interferon Tipo I/imunologia , Vacina contra Sarampo/imunologia , Vírus do Sarampo/fisiologia , Sarampo/virologia , Proteína Cofatora de Membrana/metabolismo , Replicação Viral , Animais , Chlorocebus aethiops , Humanos , Sarampo/prevenção & controle , Vírus do Sarampo/imunologia , Proteína Cofatora de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade da Espécie , Vacinas Atenuadas/imunologia , Células Vero
17.
Sci Rep ; 8(1): 12973, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154478

RESUMO

Autophagy plays a major role in defending against oxidative stress in respiratory epithelial cells. The complement regulatory protein CD46 can enhance autophagy and decrease local complement activation at sites of inflammation. This study investigated the mechanism by which CD46 protects against oxidative stress-mediated apoptosis in respiratory epithelium in asthmatic patients. Nasal mucosa samples were obtained from 60 adults with mild asthma who received turbinectomy and 30 controls. A decreased expression of CD46 and increased apoptosis were noted in the damaged nasal epithelium from the asthmatic patients. Primary epithelial cells cultured with Dermatophagoides pteronyssinus 2 showed decreased CD46 and increased cleaved CASPASE-3A expressions. Crosslinking CD46 mAb could induce the formation of autophagosomes and LC3-II expression in primary epithelial cells. CD46 engagement could induce autophagy against hydrogen peroxide-induced epithelial cell death, whereas the autophagy inhibitor 3-methyladenine decreased this effect. In addition, CD46 engagement decreased the expressions of PRO-IL-1ß and NLRP3, enhanced the expression of scaffold protein GOPC, and diminished hydrogen peroxide-induced 8-OHdG, IL-1ß and IL-6 production. Silencing ATG5 in human lung epithelial A549 cells decreased CD46-activated autophagy with LC3-II. CD46 induced autophagy and decreased the oxidative stress-mediated apoptosis of respiratory epithelium, and this may offer a new therapeutic strategy to treat asthma.


Assuntos
Apoptose , Asma/metabolismo , Autofagia , Células Epiteliais/metabolismo , Proteína Cofatora de Membrana/metabolismo , Mucosa Nasal/metabolismo , Estresse Oxidativo , Células A549 , Adulto , Antígenos de Dermatophagoides , Proteínas de Artrópodes , Asma/patologia , Caspase 3 , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Mucosa Nasal/patologia
18.
Neuro Oncol ; 20(12): 1606-1615, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-29912438

RESUMO

Background: Oncolytic measles virus (MV) is effective in xenograft models of many tumor types in immune-compromised mice. However, no murine cell line exists that is tumorigenic, grows in immune-competent mice, and is killed by MV. The lack of such a model prevents an examination of the effect of the immune system on MV oncotherapy. Methods: Cerebellar stem cells from human CD46-transgenic immunocompetent mice were transduced to express Sendai virus C-protein, murine C-Myc, and Gfi1b proteins. The resultant cells were injected into the brain of NSG mice, and a cell line, called CSCG, was prepared from the resulting tumor. Results: CSCG cells are highly proliferative, and express stem cell markers. These cells are permissive for replication of MV and are killed by the virus in a dose- and time-dependent manner. CSCG cells form aggressive tumors that morphologically resemble medulloblastoma when injected into the brains of immune-competent mice. On the molecular level, CSCG tumors overexpress natriuretic peptide receptor 3 and gamma-aminobutyric acid type A receptor alpha 5, markers of Group 3 medulloblastoma. A single intratumoral injection of MV‒green fluorescent protein resulted in complete tumor regression and prolonged survival of animals compared with treatments with phosphate buffered saline (P = 0.0018) or heat-inactivated MV (P = 0.0027). Conclusions: This immune-competent model provides the first platform to test therapeutic regimens of oncolytic MV for Group 3 medulloblastoma in the presence of anti-measles immunity. The strategy presented here can be used to make MV-sensitive murine models of any human tumor for which the driving mutations are known.


Assuntos
Neoplasias Cerebelares/terapia , Modelos Animais de Doenças , Imunocompetência , Vírus do Sarampo/genética , Meduloblastoma/terapia , Terapia Viral Oncolítica , Animais , Neoplasias Cerebelares/imunologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/virologia , Humanos , Sarampo/virologia , Meduloblastoma/imunologia , Meduloblastoma/metabolismo , Meduloblastoma/virologia , Proteína Cofatora de Membrana/genética , Proteína Cofatora de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Tumorais Cultivadas , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Immunol Lett ; 200: 26-32, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29902483

RESUMO

Similar to CD4+ T cells, precursor CD8+ T cells are thought to depend on a co-stimulatory signal through CD28 for proliferation and differentiation into effector cells. CD46 is another co-stimulatory receptor that promotes differentiation of CD4+ T-helper cells type 1 (Th1 cells) into a regulatory phenotype with a switch from IFN-γ towards IL-10-secretion over time. Whether CD46 exerts a similar function on CD8+ T cells remains to be fully elucidated. Here, we demonstrate that CD46 co-stimulation induced secretion of IFN-γ as well as expansion of IFN-γ-secreting CD8+ T cells. In contrast to CD46 co-stimulation of CD4+ T cells, CD8+ T cells did not differentiate into a regulatory IL-10-secreting phenotype. This demonstrates that CD46 is a co-stimulatory receptor on CD8+ T cells, and that it exerts separate functions during CD4+ and CD8+ T-cell differentiation.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interferon gama/biossíntese , Ativação Linfocitária/imunologia , Proteína Cofatora de Membrana/metabolismo , Biomarcadores , Citocinas/biossíntese , Humanos , Imunofenotipagem , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
20.
J Immunother Cancer ; 6(1): 55, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29898782

RESUMO

BACKGROUND: Oncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses. METHODS: Transgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines. RESULTS: Our results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication. CONCLUSION: Together, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.


Assuntos
Adenoviridae/patogenicidade , Proteína Cofatora de Membrana/metabolismo , Terapia Viral Oncolítica/métodos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos
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