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1.
Int J Mol Sci ; 22(5)2021 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-33802281

RESUMO

Many immuno-therapeutic strategies are currently being developed to fight cancer. In this scenario, oncolytic adenoviruses (Onc.Ads) have an interesting role for their peculiar tumor selectivity, safety, and transgene-delivery capability. The major strength of the Onc.Ads is the extraordinary immunogenicity that leads to a strong T-cell response, which, together with the possibility of the delivery of a therapeutic transgene, could be more effective than current strategies. In this review, we travel in the adenovirus (Ads) and Onc.Ads world, focusing on a variety of strategies that can enhance Onc.Ads antitumoral efficacy, passing through tumor microenvironment modulation. Onc.Ads-based therapeutic strategies constitute additional weapons in the fight against cancer and appear to potentiate conventional and immune checkpoint inhibitors (ICIs)-based therapies leading to a promising scenario.


Assuntos
Adenoviridae/genética , Neoplasias/terapia , Neoplasias/virologia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Animais , Terapia Genética/métodos , Humanos , Linfócitos T/virologia , Microambiente Tumoral/genética
2.
Genes (Basel) ; 12(5)2021 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923155

RESUMO

Single-cell RNA sequencing of the bronchoalveolar lavage fluid (BALF) samples from COVID-19 patients has enabled us to examine gene expression changes of human tissue in response to the SARS-CoV-2 virus infection. However, the underlying mechanisms of COVID-19 pathogenesis at single-cell resolution, its transcriptional drivers, and dynamics require further investigation. In this study, we applied machine learning algorithms to infer the trajectories of cellular changes and identify their transcriptional programs. Our study generated cellular trajectories that show the COVID-19 pathogenesis of healthy-to-moderate and healthy-to-severe on macrophages and T cells, and we observed more diverse trajectories in macrophages compared to T cells. Furthermore, our deep-learning algorithm DrivAER identified several pathways (e.g., xenobiotic pathway and complement pathway) and transcription factors (e.g., MITF and GATA3) that could be potential drivers of the transcriptomic changes for COVID-19 pathogenesis and the markers of the COVID-19 severity. Moreover, macrophages-related functions corresponded more to the disease severity compared to T cells-related functions. Our findings more proficiently dissected the transcriptomic changes leading to the severity of a COVID-19 infection.


Assuntos
Líquido da Lavagem Broncoalveolar/virologia , /patologia , Macrófagos , Linfócitos T , Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Aprendizado de Máquina , Macrófagos/fisiologia , Macrófagos/virologia , Análise de Sequência de RNA/métodos , Análise de Célula Única , Linfócitos T/fisiologia , Linfócitos T/virologia
3.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652764

RESUMO

Infection of hosts by morbilliviruses is facilitated by the interaction between viral hemagglutinin (H-protein) and the signaling lymphocytic activation molecule (SLAM). Recently, the functional importance of the n-terminal region of human SLAM as a measles virus receptor was demonstrated. However, the functional roles of this region in the infection process by other morbilliviruses and host range determination remain unknown, partly because this region is highly flexible, which has hampered accurate structure determination of this region by X-ray crystallography. In this study, we analyzed the interaction between the H-protein from canine distemper virus (CDV-H) and SLAMs by a computational chemistry approach. Molecular dynamics simulations and fragment molecular orbital analysis demonstrated that the unique His28 in the N-terminal region of SLAM from Macaca is a key determinant that enables the formation of a stable interaction with CDV-H, providing a basis for CDV infection in Macaca. The computational chemistry approach presented should enable the determination of molecular interactions involving regions of proteins that are difficult to predict from crystal structures because of their high flexibility.


Assuntos
Vírus da Cinomose Canina/genética , Cinomose/genética , Doenças do Cão/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Animais , Química Computacional , Cinomose/virologia , Vírus da Cinomose Canina/patogenicidade , Doenças do Cão/virologia , Cães , Humanos , Macaca/virologia , Mutação Puntual/genética , Ligação Proteica/genética , Receptores Virais/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/química , Família de Moléculas de Sinalização da Ativação Linfocitária/ultraestrutura , Especificidade da Espécie , Linfócitos T/virologia
4.
Front Immunol ; 12: 637651, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767706

RESUMO

As COVID-19 cases continue to rise, it is imperative to learn more about antibodies and T-cells produced against the causative virus, SARS-CoV-2, in order to guide the rapid development of therapies and vaccines. While much of the current antibody and vaccine research focuses on the receptor-binding domain of S1, a less-recognized opportunity is to harness the potential benefits of the more conserved S2 subunit. Similarities between the spike proteins of both SARS-CoV-2 and HIV-1 warrant exploring S2. Possible benefits of employing S2 in therapies and vaccines include the structural conservation of S2, extant cross-reactive neutralizing antibodies in populations (due to prior exposure to common cold coronaviruses), the steric neutralization potential of antibodies against S2, and the stronger memory B-cell and T-cell responses. More research is necessary on the effect of glycans on the accessibility and stability of S2, SARS-CoV-2 mutants that may affect infectivity, the neutralization potential of antibodies produced by memory B-cells, cross-reactive T-cell responses, antibody-dependent enhancement, and antigen competition. This perspective aims to highlight the evidence for the potential advantages of using S2 as a target of therapy or vaccine design.


Assuntos
/uso terapêutico , /imunologia , Glicoproteína da Espícula de Coronavírus/uso terapêutico , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , /virologia , Reações Cruzadas , Epitopos , Interações Hospedeiro-Patógeno , Humanos , Imunogenicidade da Vacina , Subunidades Proteicas , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/uso terapêutico
5.
Front Immunol ; 12: 655934, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777054

RESUMO

COVID-19 manifests with a wide diversity of clinical phenotypes characterized by dysfunctional and exaggerated host immune responses. Many results have been described on the status of the immune system of patients infected with SARS-CoV-2, but there are still aspects that have not been fully characterized or understood. In this study, we have analyzed a cohort of patients with mild, moderate and severe disease. We performed flow cytometric studies and correlated the data with the clinical characteristics and clinical laboratory values of the patients. Both conventional and unsupervised data analyses concluded that patients with severe disease are characterized, among others, by a higher state of activation in all T cell subsets (CD4, CD8, double negative and T follicular helper cells), higher expression of perforin and granzyme B in cytotoxic cells, expansion of adaptive NK cells and the accumulation of activated and immature dysfunctional monocytes which are identified by a low expression of HLA-DR and an intriguing shift in the expression pattern of CD300 receptors. More importantly, correlation analysis showed a strong association between the alterations in the immune cells and the clinical signs of severity. These results indicate that patients with severe COVID-19 have a broad perturbation of their immune system, and they will help to understand the immunopathogenesis of COVID-19.


Assuntos
/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Monócitos/imunologia , Receptores Imunológicos/sangue , Linfócitos T/imunologia , Idoso , /diagnóstico , Estudos de Casos e Controles , Estudos Transversais , Feminino , Citometria de Fluxo , Interações Hospedeiro-Patógeno , Humanos , Imunofenotipagem , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/virologia , Fenótipo , Índice de Gravidade de Doença , Linfócitos T/metabolismo , Linfócitos T/virologia
6.
Viruses ; 13(2)2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540662

RESUMO

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.


Assuntos
Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Linfócitos T/virologia , Linhagem Celular , Humanos , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Montagem de Vírus , Replicação Viral
7.
BMC Infect Dis ; 21(1): 56, 2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33435880

RESUMO

BACKGROUND: Hepatitis B virus (HBV) infection is a high-risk factor of hepatocellular carcinoma (HCC). Cellular immune responses are essential for HCC development, and the CD4+ and CD8+ T subtypes are identified as the primary anti-tumor immune cells. In the study, we investigated the effect and mechanism of amygdalin in the cellular immune response in HBV-related HCC and HCC progression. METHODS: The cell proliferation was examined by MTT analysis. Cells metastasis ability was detected by Invasion and migration assays. Quantification of apoptotic cells was performed with Flow cytometer assay. The protein levels of p-STAT3, STAT3, p-JAK2, JAK2, caspase-3, cleaved caspase-3 were detected by performing immunoblotting assays. RESULTS: We demonstrate that amygdalin treatment could rescue the HBV-T cell viability and IFN-γ and TNF-αproduction. In HBV-T cells, the MFI levels of CD8+ are lower than that in NC-T cells. Moreover, the phosphorylation levels of STAT3 and JAK2 are higher in HBV-T cells, compared to those in NC-T cells, and then reduced by amygdalin treatment. Co-culture with HBV-T cells could reduce IFN-γ and TNF-α, production while increase IL-6 and IL-10 production in HepG2.2.15 cells; these alterations could be partially reversed by amygdalin pretreatment. Finally, co-culture with HBV-T cells significantly promoted the cell viability, inhibited the apoptosis, and promoted the migration of HepG2.2.15 cells, and these alterations could be partially reversed by amygdalin treatment. CONCLUSION: Our findings provide a rationale for further studies on the functions and mechanism of amygdalin inhibiting HBV-related HCC cell proliferation, invasion, and migration via T cell-mediated tumor immunity.


Assuntos
Amigdalina/farmacologia , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/virologia , Progressão da Doença , Hepatite B/complicações , Janus Quinase 2/metabolismo , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/virologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Idoso , Amigdalina/metabolismo , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Células Hep G2 , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo , Linfócitos T/virologia
8.
PLoS One ; 16(1): e0245532, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33493185

RESUMO

BACKGROUND: Understanding the T cell response to SARS-CoV-2 is critical to vaccine development, epidemiological surveillance and disease control strategies. This systematic review critically evaluates and synthesises the relevant peer-reviewed and pre-print literature published from 01/01/2020-26/06/2020. METHODS: For this systematic review, keyword-structured literature searches were carried out in MEDLINE, Embase and COVID-19 Primer. Papers were independently screened by two researchers, with arbitration of disagreements by a third researcher. Data were independently extracted into a pre-designed Excel template and studies critically appraised using a modified version of the MetaQAT tool, with resolution of disagreements by consensus. Findings were narratively synthesised. RESULTS: 61 articles were included. 55 (90%) studies used observational designs, 50 (82%) involved hospitalised patients with higher acuity illness, and the majority had important limitations. Symptomatic adult COVID-19 cases consistently show peripheral T cell lymphopenia, which positively correlates with increased disease severity, duration of RNA positivity, and non-survival; while asymptomatic and paediatric cases display preserved counts. People with severe or critical disease generally develop more robust, virus-specific T cell responses. T cell memory and effector function has been demonstrated against multiple viral epitopes, and, cross-reactive T cell responses have been demonstrated in unexposed and uninfected adults, but the significance for protection and susceptibility, respectively, remains unclear. CONCLUSION: A complex pattern of T cell response to SARS-CoV-2 infection has been demonstrated, but inferences regarding population level immunity are hampered by significant methodological limitations and heterogeneity between studies, as well as a striking lack of research in asymptomatic or pauci-symptomatic individuals. In contrast to antibody responses, population-level surveillance of the T cell response is unlikely to be feasible in the near term. Focused evaluation in specific sub-groups, including vaccine recipients, should be prioritised.


Assuntos
/patologia , Linfopenia/patologia , Linfócitos T/patologia , /complicações , /virologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Celular , Linfopenia/etiologia , Linfopenia/imunologia , Linfopenia/virologia , Linfócitos T/imunologia , Linfócitos T/virologia
9.
Cancer Cell ; 39(2): 257-275.e6, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33476581

RESUMO

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.


Assuntos
/imunologia , Neoplasias/imunologia , Neoplasias/virologia , Síndrome Respiratória Aguda Grave/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , /mortalidade , Feminino , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/terapia , Neoplasias Hematológicas/virologia , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Neoplasias/mortalidade , Neoplasias/terapia , Síndrome Respiratória Aguda Grave/etiologia , Síndrome Respiratória Aguda Grave/mortalidade , Síndrome Respiratória Aguda Grave/virologia , Linfócitos T/virologia , Eliminação de Partículas Virais , Adulto Jovem
10.
Viruses ; 13(1)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478139

RESUMO

The first step of cellular entry for the human immunodeficiency virus type-1 (HIV-1) occurs through the binding of its envelope protein (Env) with the plasma membrane receptor CD4 and co-receptor CCR5 or CXCR4 on susceptible cells, primarily CD4+ T cells and macrophages. Although there is considerable knowledge of the molecular interactions between Env and host cell receptors that lead to successful fusion, the precise way in which HIV-1 receptors redistribute to sites of virus binding at the nanoscale remains unknown. Here, we quantitatively examine changes in the nanoscale organisation of CD4 on the surface of CD4+ T cells following HIV-1 binding. Using single-molecule super-resolution imaging, we show that CD4 molecules are distributed mostly as either individual molecules or small clusters of up to 4 molecules. Following virus binding, we observe a local 3-to-10-fold increase in cluster diameter and molecule number for virus-associated CD4 clusters. Moreover, a similar but smaller magnitude reorganisation of CD4 was also observed with recombinant gp120. For one of the first times, our results quantify the nanoscale CD4 reorganisation triggered by HIV-1 on host CD4+ T cells. Our quantitative approach provides a robust methodology for characterising the nanoscale organisation of plasma membrane receptors in general with the potential to link spatial organisation to function.


Assuntos
Antígenos CD4/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virologia , HIV-1/fisiologia , Imagem Individual de Molécula/métodos , Linfócitos T/metabolismo , Linfócitos T/virologia , Ligação Viral , Algoritmos , Anticorpos Monoclonais , Linhagem Celular , Interpretação Estatística de Dados , Proteína gp120 do Envelope de HIV/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Processamento de Imagem Assistida por Computador , Ligação Proteica , Receptores CCR5/metabolismo , Receptores de HIV/metabolismo
11.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33464307

RESUMO

Virus-specific T cells play essential roles in protection against multiple virus infections, including SARS-CoV and MERS-CoV. While SARS-CoV-2-specific T cells have been identified in COVID-19 patients, their role in the protection of SARS-CoV-2-infected mice is not established. Here, using mice sensitized for infection with SARS-CoV-2 by transduction with an adenovirus expressing the human receptor (Ad5-hACE2), we identified SARS-CoV-2-specific T cell epitopes recognized by CD4+ and CD8+ T cells in BALB/c and C57BL/6 mice. Virus-specific T cells were polyfunctional and were able to lyse target cells in vivo. Further, type I interferon pathway was proved to be critical for generating optimal antiviral T cell responses after SARS-CoV-2 infection. T cell vaccination alone partially protected SARS-CoV-2-infected mice from severe disease. In addition, the results demonstrated cross-reactive T cell responses between SARS-CoV and SARS-CoV-2, but not MERS-CoV, in mice. Understanding the role of the T cell response will guide immunopathogenesis studies of COVID-19 and vaccine design and validation.


Assuntos
/imunologia , Epitopos de Linfócito T/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Linfócitos T/imunologia , Linfócitos T/virologia , /genética , Animais , Anticorpos Neutralizantes/sangue , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Chlorocebus aethiops , Reações Cruzadas , Mapeamento de Epitopos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Vírus da SARS/imunologia , /patogenicidade , Células Vero
12.
Chem Commun (Camb) ; 57(4): 504-507, 2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33331360

RESUMO

A novel STING agonist, CDGSF, ipsilaterally modified with phosphorothioate and fluorine, was synthesized. The phosphorothioate in CDGSF might be a site for covalent conjugation. Injection of CDGSF generated an immunogenic ("hot") tumor microenvironment to suppress melanoma, more efficiently than dithio CDG. In particular, immunization with SARS-CoV-2 spike protein using CDGSF as an adjuvant elicited an exceptionally high antibody titer and a robust T cell response, overcoming the drawbacks of aluminum hydroxide. These results highlighted the therapeutic potential of CDGSF for cancer immunotherapy and the adjuvant potential of the STING agonist in the SARS-CoV-2 vaccine for the first time.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , /prevenção & controle , Melanoma Experimental/tratamento farmacológico , Proteínas de Membrana/agonistas , Nucleotídeos Cíclicos/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Adjuvantes Imunológicos/síntese química , Hidróxido de Alumínio/administração & dosagem , Hidróxido de Alumínio/química , Animais , Anticorpos Antivirais/biossíntese , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/virologia , /virologia , ELISPOT , Humanos , Imunoterapia/métodos , Interferon gama/biossíntese , Melanoma Experimental/imunologia , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Nucleotídeos Cíclicos/síntese química , /imunologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Vacinação/métodos
13.
Immunology ; 162(1): 30-43, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32935333

RESUMO

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2, a novel coronavirus strain. Some studies suggest that COVID-19 could be an immune-related disease, and failure of effective immune responses in initial stages of viral infection could contribute to systemic inflammation and tissue damage, leading to worse disease outcomes. T cells can act as a double-edge sword with both pro- and anti-roles in the progression of COVID-19. Thus, better understanding of their roles in immune responses to SARS-CoV-2 infection is crucial. T cells primarily react to the spike protein on the coronavirus to initiate antiviral immunity; however, T-cell responses can be suboptimal, impaired or excessive in severe COVID-19 patients. This review focuses on the multifaceted roles of T cells in COVID-19 pathogenesis and rationalizes their significance in eliciting appropriate antiviral immune responses in COVID-19 patients and unexposed individuals. In addition, we summarize the potential therapeutic approaches related to T cells to treat COVID-19 patients. These include adoptive T-cell therapies, vaccines activating T-cell responses, recombinant cytokines, Th1 activators and Th17 blockers, and potential utilization of immune checkpoint inhibitors alone or in combination with anti-inflammatory drugs to improve antiviral T-cell responses against SARS-CoV-2.


Assuntos
/imunologia , Imunidade Celular , Imunoterapia , Pulmão/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Antivirais/uso terapêutico , /virologia , Interações Hospedeiro-Patógeno , Humanos , Imunidade Celular/efeitos dos fármacos , Fatores Imunológicos/uso terapêutico , Pulmão/efeitos dos fármacos , Pulmão/virologia , /patogenicidade , Linfócitos T/efeitos dos fármacos , Linfócitos T/transplante , Linfócitos T/virologia
14.
Methods Mol Biol ; 2167: 225-252, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712923

RESUMO

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18 base pairs (bp) stem and a 3-5 nucleotides (nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.


Assuntos
Proteínas Argonauta/genética , Inativação Gênica , Infecções por HIV/genética , HIV-1/genética , Vírus Delta da Hepatite/genética , RNA Catalítico/genética , RNA Guia/genética , RNA Interferente Pequeno/genética , Proteínas Argonauta/metabolismo , Northern Blotting , Clonagem Molecular/métodos , Ensaios Enzimáticos/métodos , Vetores Genéticos , Células HEK293 , HIV-1/metabolismo , Humanos , Sequências Repetidas Invertidas/genética , Luciferases/metabolismo , Interferência de RNA , RNA Catalítico/metabolismo , RNA Guia/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/virologia , Transfecção/métodos
15.
PLoS Pathog ; 16(12): e1009163, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33326500

RESUMO

The novel human coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Critical to the rapid evaluation of vaccines and antivirals against SARS-CoV-2 is the development of tractable animal models to understand the adaptive immune response to the virus. To this end, the use of common laboratory strains of mice is hindered by significant divergence of the angiotensin-converting enzyme 2 (ACE2), which is the receptor required for entry of SARS-CoV-2. In the current study, we designed and utilized an mRNA-based transfection system to induce expression of the hACE2 receptor in order to confer entry of SARS-CoV-2 in otherwise non-permissive cells. By employing this expression system in an in vivo setting, we were able to interrogate the adaptive immune response to SARS-CoV-2 in type 1 interferon receptor deficient mice. In doing so, we showed that the T cell response to SARS-CoV-2 is enhanced when hACE2 is expressed during infection. Moreover, we demonstrated that these responses are preserved in memory and are boosted upon secondary infection. Importantly, using this system, we functionally identified the CD4+ and CD8+ structural peptide epitopes targeted during SARS-CoV-2 infection in H2b restricted mice and confirmed their existence in an established model of SARS-CoV-2 pathogenesis. We demonstrated that, identical to what has been seen in humans, the antigen-specific CD8+ T cells in mice primarily target peptides of the spike and membrane proteins, while the antigen-specific CD4+ T cells target peptides of the nucleocapsid, membrane, and spike proteins. As the focus of the immune response in mice is highly similar to that of the humans, the identification of functional murine SARS-CoV-2-specific T cell epitopes provided in this study will be critical for evaluation of vaccine efficacy in murine models of SARS-CoV-2 infection.


Assuntos
Imunidade Adaptativa/imunologia , /imunologia , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Replicação Viral , /genética , Animais , /virologia , Chlorocebus aethiops , Epitopos de Linfócito T/imunologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , Receptor de Interferon alfa e beta/fisiologia , Linfócitos T/virologia , Células Vero
16.
Viruses ; 13(1)2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33375604

RESUMO

Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-ß treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.


Assuntos
Edição de Genes , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Tropismo Viral/genética , Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/genética , Humanos , Células Jurkat , RNA Guia , Linfócitos T/imunologia
17.
Proc Natl Acad Sci U S A ; 117(52): 32880-32882, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318172

RESUMO

In vivo clonal expansion of HIV-infected T cells is an important mechanism of viral persistence. In some cases, clonal expansion is driven by HIV proviral DNA integrated into one of a handful of genes. To investigate this phenomenon in vitro, we infected primary CD4+ T cells with an HIV construct expressing GFP and, after nearly 2 mo of culture and multiple rounds of activation, analyzed the resulting integration site distribution. In each of three replicates from each of two donors, we detected large clusters of integration sites with multiple breakpoints, implying clonal selection. These clusters all mapped to a narrow region within the STAT3 gene. The presence of hybrid transcripts splicing HIV to STAT3 sequences supports a model of LTR-driven STAT3 overexpression as a driver of preferential growth. Thus, HIV integration patterns linked to selective T cell outgrowth can be reproduced in cell culture. The single report of an HIV provirus in a case of AIDS-associated B-cell lymphoma with an HIV provirus in the same part of STAT3 also has implications for HIV-induced malignancy.


Assuntos
Proliferação de Células , HIV/fisiologia , Provírus/fisiologia , Linfócitos T/virologia , Integração Viral , Células Cultivadas , Evolução Clonal , DNA Viral/genética , HIV/genética , Humanos , Provírus/genética , Fator de Transcrição STAT3/genética , Linfócitos T/fisiologia
18.
Nat Commun ; 11(1): 5660, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168830

RESUMO

Human endogenous retroviruses (HERV) form a substantial part of the human genome, but mostly remain transcriptionally silent under strict epigenetic regulation, yet can potentially be reactivated by malignant transformation or epigenetic therapies. Here, we evaluate the potential for T cell recognition of HERV elements in myeloid malignancies by mapping transcribed HERV genes and generating a library of 1169 potential antigenic HERV-derived peptides predicted for presentation by 4 HLA class I molecules. Using DNA barcode-labeled MHC-I multimers, we find CD8+ T cell populations recognizing 29 HERV-derived peptides representing 18 different HERV loci, of which HERVH-5, HERVW-1, and HERVE-3 have more profound responses; such HERV-specific T cells are present in 17 of the 34 patients, but less frequently in healthy donors. Transcriptomic analyses reveal enhanced transcription of the HERVs in patients; meanwhile DNA-demethylating therapy causes a small and heterogeneous enhancement in HERV transcription without altering T cell recognition. Our study thus uncovers T cell recognition of HERVs in myeloid malignancies, thereby implicating HERVs as potential targets for immunotherapeutic therapies.


Assuntos
Retrovirus Endógenos/genética , Neoplasias Hematológicas/virologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Linfócitos T CD8-Positivos , Epigênese Genética , Epitopos de Linfócito T , Perfilação da Expressão Gênica , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Imunoterapia , Monitorização Imunológica , Células Mieloides , Neoplasias
19.
PLoS Pathog ; 16(9): e1008879, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32997728

RESUMO

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24-45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26-37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells.


Assuntos
Moléculas de Adesão Celular , Adesões Focais , Produtos do Gene gag , Vírus Linfotrópico T Tipo 1 Humano , Proteínas dos Microfilamentos , Fosfoproteínas , Linfócitos T , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Adesões Focais/química , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/virologia , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Células Jurkat , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Domínios Proteicos , Linfócitos T/química , Linfócitos T/metabolismo , Linfócitos T/virologia
20.
Proc Natl Acad Sci U S A ; 117(40): 24964-24973, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958663

RESUMO

Influenza A virus (IAV) infection during pregnancy causes severe maternal and perinatal complications, despite a lack of vertical transmission of IAV across the placenta. Here, we demonstrate a significant alteration in the maternal vascular landscape that underpins the maternal and downstream fetal pathology to IAV infection in mice. In IAV infection of nonpregnant mice, the local lung inflammatory response was contained to the lungs and was self-resolving, whereas in pregnant mice, virus dissemination to major maternal blood vessels, including the aorta, resulted in a peripheral "vascular storm," with elevated proinflammatory and antiviral mediators and the influx of Ly6Clow and Ly6Chigh monocytes, plus neutrophils and T cells. This vascular storm was associated with elevated levels of the adhesion molecules ICAM and VCAM and the pattern-recognition receptors TLR7 and TLR9 in the vascular wall, resulting in profound vascular dysfunction. The sequalae of this IAV-driven vascular storm included placental growth retardation and intrauterine growth restriction, evidence of placental and fetal brain hypoxia, and increased circulating cell free fetal DNA and soluble Flt1. In contrast, IAV infection in nonpregnant mice caused no obvious alterations in endothelial function or vascular inflammation. Therefore, IAV infection during pregnancy drives a significant systemic vascular alteration in pregnant dams, which likely suppresses critical blood flow to the placenta and fetus. This study in mice provides a fundamental mechanistic insight and a paradigm into how an immune response to a respiratory virus, such as IAV, is likely to specifically drive maternal and fetal pathologies during pregnancy.


Assuntos
Imunidade Adaptativa/genética , Imunidade Inata/genética , Inflamação/genética , Vírus da Influenza A/genética , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Feminino , Feto/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Vírus da Influenza A/patogenicidade , Influenza Humana/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Glicoproteínas de Membrana/genética , Camundongos , Monócitos/metabolismo , Monócitos/patologia , Placenta/irrigação sanguínea , Placenta/imunologia , Placenta/virologia , Gravidez , Linfócitos T/imunologia , Linfócitos T/virologia , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética
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