RESUMO
Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.
Assuntos
Células Dendríticas , Macaca mulatta , Vacinas contra a SAIDS , Vírus da Imunodeficiência Símia , Animais , Vírus da Imunodeficiência Símia/imunologia , Células Dendríticas/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Proteínas de Membrana/imunologia , Proteínas de Membrana/genética , Tirosina Quinase 3 Semelhante a fms/imunologia , Tirosina Quinase 3 Semelhante a fms/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vetores Genéticos , Imunogenicidade da Vacina , Linfócitos T CD8-Positivos/imunologiaRESUMO
Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.
Assuntos
Alanina , Antivirais , Ebolavirus , Doença pelo Vírus Ebola , Nucleosídeos , Pró-Fármacos , Animais , Administração Oral , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/prevenção & controle , Macaca fascicularis , Nucleosídeos/administração & dosagem , Nucleosídeos/farmacologia , Monofosfato de Adenosina/administração & dosagem , Monofosfato de Adenosina/farmacologia , Alanina/administração & dosagem , Alanina/análogos & derivados , Alanina/farmacologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Antivirais/administração & dosagem , Antivirais/farmacologiaRESUMO
Developing an intervention that results in virologic control following discontinuation of antiretroviral therapy (ART) is a major objective of HIV-1 cure research. In this study, we investigated the therapeutic efficacy of a vaccine consisting of adenovirus serotype 26 (Ad26) and modified vaccinia Ankara (MVA) with or without an SIV Envelope (Env) gp140 protein with alum adjuvant in combination with the TLR7 agonist vesatolimod (GS-9620) in 36 ART-suppressed, SIVmac251-infected rhesus macaques. Ad26/MVA therapeutic vaccination led to robust humoral and cellular immune responses, and the Env protein boost increased antibody responses. Following discontinuation of ART, virologic control was observed in 5/12 animals in each vaccine group, compared with 0/12 animals in the sham control group. These data demonstrate therapeutic efficacy of Ad26/MVA vaccination with vesatolimod but no clear additional benefit of adding an Env protein boost. SIV-specific cellular immune responses correlated with virologic control. Our findings show partial efficacy of therapeutic vaccination following ART discontinuation in SIV-infected rhesus macaques.
RESUMO
Globally, 1.8 million children are living with HIV-1. While antiretroviral therapy (ART) has improved disease outcomes, it does not eliminate the latent HIV-1 reservoir. Interventions to delay or prevent viral rebound in the absence of ART would be highly beneficial for HIV-1-infected children who now must remain on daily ART throughout their lifespan. Here, we evaluated therapeutic Ad48-SIV prime, MVA-SIV boost immunization in combination with the TLR-7 agonist GS-986 in rhesus macaque (RM) infants orally infected with SIVmac251 at 4 weeks of age and treated with a triple ART regimen beginning 4 weeks after infection. We hypothesized immunization would enhance SIV-specific T cell responses during ART-mediated suppression of viremia. Compared to controls, vaccinated infants had greater magnitude SIV-specific T cell responses (mean of 3475 vs 69 IFN-γ spot forming cells (SFC) per 106 PBMCs, respectively, P = 0.01) with enhanced breadth of epitope recognition and increased CD8+ and CD4+ T cell polyfunctionality (P = 0.004 and P = 0.005, respectively). Additionally, SIV-specific gp120 antibodies against challenge and vaccine virus strains were significantly elevated following MVA boost (P = 0.02 and P < 0.001, respectively). GS-986 led to expected immune stimulation demonstrated by activation of monocytes and T cells 24 hours post-dose. Despite the vaccine-induced immune responses, levels of SIV DNA in peripheral and lymph node CD4+ T cells were not significantly different from controls and a similar time to viral rebound and viral load set point were observed following ART interruption in both groups. We demonstrate infant RMs mount a robust immunological response to this immunization, but vaccination alone was not sufficient to impact viral reservoir size or modulate rebound dynamics following ART release. Our findings hold promise for therapeutic vaccination as a part of a combination cure approach in children and highlight the importance of a pediatric model to evaluate HIV-1 cure interventions in this unique setting of immune development.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/imunologia , Receptor 7 Toll-Like/agonistas , Vacinação/métodos , Viremia/tratamento farmacológico , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Linfócitos T CD4-Positivos/virologia , Feminino , Vetores Genéticos , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Carga Viral , Viremia/imunologia , Viremia/virologia , Replicação ViralRESUMO
BACKGROUND: Vesatolimod (VES; GS-9620) is a Toll-like receptor 7 (TLR7) agonist that directly activates human plasmacytoid dendritic cells (pDCs) and B lymphocytes resulting in direct and indirect production of cytokines and immune activation. VES is being evaluated in HIV-1-infected people as part of an HIV remission strategy. Here we investigated the potential of VES to trigger indirect activation of HIV-specific CD8+ T-cells using immune cell cultures derived from HIV+ donors. METHODS: Peripheral blood mononuclear cell (PBMC) cultures derived from HIV+ donors virologically suppressed on stable antiretroviral therapy (n=31) were isolated and treated with VES or vehicle for 24 h. Cells were stained with surface and intracellular fluorescent conjugated antibodies and HIV-specific pentamers, and analysed by flow cytometry. RESULTS: Treatment of PBMCs with VES resulted in all 31 donors demonstrating a concentration dependent increase in CD8+ T-cell activation (CD69+) of up to 88%. Of these donors, 20 of 31 donors displayed a concentration-dependent increase in HIV-specific CD8+ T-cell activation due to VES with a maximum of 20.8%. Intracellular staining was performed in a subset of donors (n=14), 5 of which displayed VES-induced activation of functional HIV-specific CD8+ T-cells as assessed by CD107a and/or tumour necrosis factor (TNF)-α upregulation. CONCLUSIONS: This study demonstrates that VES treatment can induce the activation of functional HIV-specific CD8+ T-cells in donor derived PBMCs. These data support the potential use of VES to activate functional HIV-specific CD8+ T-cells as part of an HIV remission strategy.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , Pteridinas/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta a Droga , Citometria de Fluxo , HIV/imunologia , Infecções por HIV/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacosRESUMO
The development of immunologic interventions that can target the viral reservoir in HIV-1-infected individuals is a major goal of HIV-1 research. However, little evidence exists that the viral reservoir can be sufficiently targeted to improve virologic control following discontinuation of antiretroviral therapy. Here we show that therapeutic vaccination with Ad26/MVA (recombinant adenovirus serotype 26 (Ad26) prime, modified vaccinia Ankara (MVA) boost) and stimulation of TLR7 (Toll-like receptor 7) improves virologic control and delays viral rebound following discontinuation of antiretroviral therapy in SIV-infected rhesus monkeys that began antiretroviral therapy during acute infection. Therapeutic vaccination with Ad26/MVA resulted in a marked increase in the magnitude and breadth of SIV-specific cellular immune responses in virologically suppressed, SIV-infected monkeys. TLR7 agonist administration led to innate immune stimulation and cellular immune activation. The combination of Ad26/MVA vaccination and TLR7 stimulation resulted in decreased levels of viral DNA in lymph nodes and peripheral blood, and improved virologic control and delayed viral rebound following discontinuation of antiretroviral therapy. The breadth of cellular immune responses correlated inversely with set point viral loads and correlated directly with time to viral rebound. These data demonstrate the potential of therapeutic vaccination combined with innate immune stimulation as a strategy aimed at a functional cure for HIV-1 infection.
Assuntos
Adenoviridae/genética , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/terapia , Vírus da Imunodeficiência Símia/imunologia , Receptor 7 Toll-Like/imunologia , Vaccinia virus/genética , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Antirretrovirais/administração & dosagem , DNA Viral/análise , DNA Viral/sangue , Feminino , Vetores Genéticos/genética , Infecções por HIV/imunologia , Infecções por HIV/terapia , Imunidade Celular , Imunidade Inata , Macaca mulatta , Masculino , RNA Viral/análise , RNA Viral/sangue , Vacinas contra a SAIDS/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/crescimento & desenvolvimento , Vírus da Imunodeficiência Símia/isolamento & purificação , Fatores de Tempo , Receptor 7 Toll-Like/genética , Carga Viral/imunologiaRESUMO
Resting CD4+ T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8+ T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8+ T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8+ T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8+ T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8+ T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam3CSK4. In contrast, we did not observe CD8+ T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist 'ALT-803', an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8+ T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8+ T-cells in HIV eradication strategies.
Assuntos
Antivirais/farmacologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Proteínas/farmacologia , Linfócitos T Citotóxicos/imunologia , Latência Viral/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão , Ativação Viral/efeitos dos fármacosRESUMO
Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50â=â4.5 nM) compared with vorinostat (VOR; EC50â=â3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50â=â10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.
Assuntos
Linfócitos T CD4-Positivos/virologia , Depsipeptídeos/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Modelos Biológicos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Depsipeptídeos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Infecções por HIV/virologia , Inibidores de Histona Desacetilases/farmacocinética , Histona Desacetilases/metabolismo , Humanos , Memória Imunológica/efeitos dos fármacos , Isoenzimas/metabolismo , MasculinoRESUMO
BACKGROUND/AIM: GS 9219 is a double prodrug of antiproliferative nucleotide analog 9-(2-Phosphonylmethoxyethyl)guanine (PMEG), with potent in vivo efficacy against various hematological malignancies. This study investigates the role of adenosine deaminase-like (ADAL) protein in the intracellular activation of GS-9219. MATERIALS AND METHODS: A cell line resistant to 9-(2-Phosphonylmethoxyethyl)-N(6)-cyclopropyl-2,6-diaminopurine (cPrPMEDAP), an intermediate metabolite of GS-9219, was generated and characterized. RESULTS: The resistant cell line was cross-resistant to cPrPMEDAP and GS-9219, due to a defect in the deamination of cPrPMEDAP to PMEG. Mutations in the ADAL gene (H286R and S180N) were identified in the resistant cells that adversely-affected its enzymatic activity. Introduction of the wild-type ADAL gene re-sensitized resistant cells to both cPrPMEDAP and GS-9219. CONCLUSION: The ADAL protein plays an essential role in the intracellular activation of GS-9219 by catalyzing the deamination of cPrPMEDAP metabolite to PMEG. Mutations affecting the activity of ADAL confer resistance to both GS-9219 and its metabolite cPrPMEDAP.
Assuntos
Adenina/análogos & derivados , Alanina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Mutação/genética , Nucleosídeo Desaminases/genética , Purinas/farmacologia , Neoplasias do Colo do Útero/genética , Adenina/farmacologia , Alanina/farmacologia , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Western Blotting , Feminino , Humanos , Dados de Sequência Molecular , Nucleosídeo Desaminases/química , Nucleosídeo Desaminases/metabolismo , Pró-Fármacos/farmacologia , Conformação Proteica , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation.
Assuntos
Ditiotreitol/farmacologia , Integrase de HIV/química , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Multimerização Proteica/efeitos dos fármacos , Integrase de HIV/metabolismo , Cinética , Ligação ProteicaRESUMO
HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors.
Assuntos
DNA Viral/metabolismo , Inibidores de Integrase de HIV/farmacocinética , Integrase de HIV/metabolismo , Catálise , Dissulfetos , Integrase de HIV/genética , HIV-1/genética , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Multimerização ProteicaRESUMO
Peptides from the N-heptad repeat region of the HIV gp41 protein can inhibit viral fusion, but their potency is limited by a low tendency to form a trimeric coiled-coil. Accordingly, stabilization of N peptides by fusion with the stable coiled-coil IZ yields nanomolar inhibitors [Eckert, D. M. & Kim, P. S. (2001) Proc. Natl. Acad. Sci. USA 98, 11187-11192]. Because the antiviral potency of IZN17 is limited by self-association equilibrium, we covalently stabilized the peptide by using interchain disulfide bonds. The resulting covalent trimer, (CCIZN17)3, has an extraordinary thermodynamic stability that translates into unprecedented antiviral potency: (CCIZN17)3 (i) inhibits fusion in a cell-cell fusion assay (IC50 = 260 pM); (ii) is the most potent fusion inhibitor described to date (IC50 = 40-380 pM) in a single-cycle infectivity assay against HIV(HXB2), HIV(NL4-3), and HIV(MN-1); (iii) efficiently neutralizes acute viral infection in peripheral blood mononuclear cells; and (iv) displays a broad antiviral profile, being able to neutralize 100% of a large panel of HIV isolates, including R5, X4, and R5/X4 strains. In all of these assays, the potency of N-peptide inhibitor (CCIZN17)3 was equal to or more than the C-peptide inhibitor in clinical use, DP178 (also known as Enfuvirtide and Fuzeon). More importantly, we show that the two inhibitors, which have different targets in gp41, synergize when used in combination. These features make (CCIZN17)3 an attractive lead to develop as an antiviral drug, alone or in combination with DP178, as well as a promising immunogen to elicit a fusion-blocking neutralizing antibody response.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Fármacos Anti-HIV/metabolismo , Fusão Celular , Dicroísmo Circular , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Enfuvirtida , Proteína gp41 do Envelope de HIV/genética , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Testes de Neutralização , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Desnaturação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Relação Estrutura-Atividade , Temperatura , TermodinâmicaRESUMO
Receptor-interacting protein (RIP) plays a critical role in tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation. However, the mechanism by which RIP mediates TNF-alpha-induced signal transduction is not fully understood. In this study, we reconstituted RIP-deficient Jurkat T cells with a fusion protein composed of full-length MEKK3 and the death domain of RIP (MEKK3-DD). In these cells, MEKK3-DD substitutes for RIP and directly associates with TRADD in TNF receptor complexes following TNF-alpha stimulation. We found that TNF-alpha-induced NF-kappaB activation was fully restored by MEKK3-DD in these cells. In contrast, expression of a fusion protein composed of NEMO, a component of the IkappaB kinase complex, and the death domain of RIP (NEMO-DD) cannot restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells. These results indicate that the role of RIP is to specifically recruit MEKK3 to the TNF-alpha receptor complex, whereas the forced recruitment of NEMO to the TNF-alpha receptor complex is insufficient for TNF-alpha-induced NF-kappaB activation. Although MEKK2 has a high degree of homology with MEKK3, MEKK2-DD, unlike MEKK3-DD, also fails to restore TNF-alpha-induced NF-kappaB activation in RIP-deficient cells, indicating that RIP-dependent recruitment of MEKK3 plays a specific role in TNF-alpha signaling.
Assuntos
MAP Quinase Quinase Quinase 3/metabolismo , NF-kappa B/metabolismo , Proteínas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Western Blotting , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Quinase I-kappa B , Células Jurkat , Modelos Biológicos , Testes de Precipitina , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas/química , Proteína Serina-Treonina Quinases de Interação com Receptores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
A series of 2',3'-dideoxy (D2) and 2',3'-didehydro-2',3'-dideoxy (D4) 5-fluorocytosine nucleosides modified with substituted benzoyl, heteroaromatic carbonyl, cycloalkylcarbonyl and alkanoyl at the N4-position were synthesized and evaluated for anti-human immunodeficiency virus type 1 (HIV-1) and anti-hepatitis B virus (HBV) activity in vitro. For most D2-nucleosides, N4-substitutions improved the anti-HIV-1 activity markedly without increasing the cytotoxicity. In the D4-nucleosides series, some of the substituents at the N4-position enhanced the anti-HIV-1 activity with a modest increase in the cytotoxicity. The most potent and selective N4-modified nucleoside for the D2-series was N4-p-iodobenzoyl-D2FC, which had a 46-fold increase in anti-HIV-1 potency in MT-2 cells compared to the parent nucleoside D-D2FC. In the D4-series, N4-p-bromobenzoyl-D4FC was 12-fold more potent in MT-2 cells compared to the parent nucleoside D-D4FC. All eight N4-p-halobenzoyl-substituted D2- and D4-nucleosides evaluated against HBV in HepAD38 cells demonstrated equal or greater potency than the two parental compounds, D-D2FC and D-D4FC. The N4-modification especially in the D2-nucleoside series containing the N4-nicotinoyl, o-nitrobenzoyl and n-butyryl showed a significant reduction in mitochondrial toxicity relative to the parent nucleoside analogue. Although the 5'-triphosphate of the parent compound (D-D4FC-TP) was formed from the N4-acyl-D4FC analogues in different cells, the levels of the 5'-triphosphate nucleotide did not correlate with the cell-derived 90% effective antiviral concentrations (EC90), suggesting that a direct interaction of the triphosphates of these N4-acyl nucleosides was involved in the antiviral activity.
Assuntos
Antivirais/farmacologia , Zalcitabina/farmacologia , Animais , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/síntese química , Antivirais/química , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Relação Dose-Resposta a Droga , HIV-1/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Células Vero , Zalcitabina/análogos & derivados , Zalcitabina/síntese química , Zalcitabina/químicaRESUMO
Helicobacter pylori-infected gastric mucosa displays a conspicuous infiltration of mononuclear cells and neutrophils. RANTES (short for "regulated upon activation, normal T cell expressed and secreted") is a chemoattractant cytokine (chemokine) important in the infiltration of T lymphocytes and monocytes. RANTES may therefore contribute to the cellular infiltrate in the H. pylori-infected gastric mucosa. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated RANTES expression. We observed that gastric epithelial cells produced RANTES upon coculture with H. pylori. In addition, H. pylori induced RANTES mRNA expression and an increase in luciferase activity in cells which were transfected with a luciferase reporter construct derived from the RANTES promoter, in gastric epithelial cells, indicating that the induction of RANTES production occurred at the transcriptional level. Induction of RANTES was dependent on an intact cag pathogenicity island. Activation of the RANTES promoter by H. pylori occurred through the action of NF-kappa B. Transfection of kinase-deficient mutants of I kappa B kinase (IKK) and NF-kappa B-inducing kinase (NIK) inhibited H. pylori-mediated RANTES activation. In contrast, tumor necrosis factor alpha- or interleukin-1/Toll-like receptor signaling molecules-such as mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1, MyD88, and interleukin-1 receptor-associated kinase-did not play a role in RANTES activation by H. pylori. Collectively, H. pylori induced NF-kappa B activation through an intracellular signaling pathway that involved IKK and NIK, leading to RANTES gene transcription. RANTES induction by H. pylori may play an important role in gastric inflammation.
Assuntos
Quimiocina CCL5/biossíntese , Helicobacter pylori/patogenicidade , NF-kappa B/fisiologia , Nitrilas , Compostos Orgânicos , Sulfonas , Antineoplásicos/farmacologia , Quimiocina CCL5/genética , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Humanos , Interleucina-1/farmacologia , Receptores de Lipopolissacarídeos/fisiologia , Glicoproteínas de Membrana/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Receptores de Superfície Celular/fisiologia , Receptores Toll-Like , Transcrição GênicaRESUMO
BACKGROUND & AIMS: Matrix metalloproteinases (MMPs), enzymes capable of degrading extracellular matrix components, are believed to be active in connective tissue remodeling associated with various physiologic processes and in pathologic conditions. The aim of this study was to analyze the molecular mechanism responsible for Helicobacter pylori-mediated MMP expression. METHODS: Expression of MMP messenger RNA and MMP activity were assessed by reverse-transcription polymerase chain reaction and zymography, respectively. Chloramphenicol acetyltransferase assay was used to monitor activation of the MMP-9 gene promoter, and electrophoretic mobility shift assay was used to explore the binding of transcription factors to this promoter. Gastric tissue samples were immunohistochemically stained for MMP-9. RESULTS: H. pylori induced MMP-9 expression in 2 gastric epithelial cell lines but had no effect on MMP-2. Induction of MMP-9 was dependent on an intact cag pathogenicity island. Activation of the MMP-9 promoter by H. pylori occurred through the action of nuclear factor kappaB. Transfection of kinase-deficient mutants of IkappaB kinase and nuclear factor kappaB-inducing kinase inhibited H. pylori-mediated activation of MMP-9. MMP-9 expression was higher in epithelial cells of H. pylori-positive tissue compared with those of H. pylori-negative tissue. CONCLUSIONS: H. pylori induced activation of nuclear factor kappaB through an intracellular signaling pathway that involved IkappaB kinase and nuclear factor kappaB-inducing kinase, leading to MMP-9 gene transcription. MMP-9 induction by H. pylori may play an important role in gastric inflammation, ulcer formation, and carcinogenesis.
Assuntos
Infecções por Helicobacter/enzimologia , Helicobacter pylori/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Células Cultivadas , Indução Enzimática , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Mucosa Gástrica/citologia , Mucosa Gástrica/microbiologia , Gastrite/enzimologia , Gastrite/microbiologia , Regulação Enzimológica da Expressão Gênica , Infecções por Helicobacter/microbiologia , Humanos , Imuno-Histoquímica , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/análise , Transdução de Sinais/fisiologia , Estômago/citologia , Estômago/microbiologia , Transcrição GênicaRESUMO
The nef gene products encoded by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus type 1 (SIV-1) increase viral loads in infected hosts and accelerate clinical progression to AIDS. Nef exhibits a spectrum of biological activities, including the ability to downregulate surface expression of CD4 and major histocompatibility complex (MHC) class I antigens, to alter the state of T-cell activation, and to enhance the infectivity of viral particles. To determine which of these in vitro functions most closely correlates with the pathogenic effects of Nef in vivo, we constructed recombinant HIV-1 NL4-3 viruses carrying mutations within the nef gene that selectively impair these functions. These mutant viruses were evaluated for pathogenic potential in severe combined immunodeficiency (SCID) mice implanted with human fetal thymus and liver (SCID-hu Thy/Liv mice), in which virus-mediated depletion of thymocytes is known to be Nef dependent. Disruption of the polyproline type II helix (Pxx)4 within Nef (required for binding of Hck and p21-activated kinase-like kinases, downregulation of MHC class I, and enhancement of HIV-1 infectivity in vitro but dispensable for CD4 downregulation) did not impair thymocyte depletion in virus-infected Thy/Liv human thymus implants. Conversely, three separate point mutations in Nef that compromised its ability to downregulate CD4 attenuated thymocyte depletion while not diminishing viral replication. These findings indicate that the functional ability of Nef to downregulate CD4 and not MHC class I downregulation, Hck or PAK binding, or (Pxx)4-associated enhancement of infectivity most closely correlates with Nef-mediated enhancement of HIV-1 pathogenicity in vivo. Nef-mediated CD4 downregulation merits consideration as a new target for the development of small-molecule inhibitors.
Assuntos
Antígenos CD4/análise , Produtos do Gene nef/fisiologia , HIV-1/patogenicidade , Alelos , Animais , Antígenos CD4/fisiologia , Linhagem Celular , Regulação para Baixo , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Camundongos , Camundongos SCID , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
Adult T-cell leukemia is caused by human T-cell leukemia virus type I (HTLV-I). The HTLV-I Tax protein is essential for clinical manifestations because it activates viral and cellular gene transcription. Tax enhances production of tumor necrosis factor-alpha (TNF-alpha), which may lead to bone and joint destruction. Because estrogens might prevent osteoporosis by repressing TNF-alpha gene transcription, we investigated whether estrogens inhibit the transcriptional effects of Tax on the TNF-alpha promoter. Tax activated the -1044, -163, and -125 TNF-alpha promoters by 9-25-fold but not the -82 promoter, demonstrating that Tax activation requires the -125 to -82 region, known as the TNF response element (TNF-RE). Three copies of the TNF-RE upstream of the minimal thymidine kinase promoter conferred a similar magnitude of activation by Tax. We demonstrated that c-Jun, NFkappaB, p50, and p65 interact with and activate the TNF-RE by using mutational analysis of the TNF-RE, Tax mutants that selectively activate NFkappaB or the cAMP-response element binding protein/activating transcription factor pathway, and gel shift assays with nuclear extracts. Estradiol markedly repressed Tax-activated transcription of the TNF-alpha gene with estrogen receptor (ER) alpha or beta. Nuclear extracts from U2OS cells stably transfected with ER(alpha) demonstrated that ERs interact with the TNF-RE. Our studies provide evidence that ERs repress Tax-activated TNF-alpha transcription by interacting with a c-Jun and NFkappaB platform on the TNF-RE. Estrogens may ameliorate bone and inflammatory joint diseases in patients infected with HTLV-I by repressing transcription of the TNF-alpha gene.
Assuntos
Estradiol/metabolismo , Regulação da Expressão Gênica , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genética , Sítios de Ligação , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Masculino , Mutação , NF-kappa B/metabolismo , Fatores de Transcrição NFATC , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores de Estrogênio/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy. Infiltration of various tissues by circulating leukemic cells is a characteristic of ATL. Matrix metalloproteinases (MMPs), which mediate the degradation of the basement membrane and extracellular matrix, play an important role in metastasis and tumor cell dissemination. The aim of this study was to explore whether expression of MMP-2 and MMP-9 was deregulated by HTLV-I infection. The data showed that HTLV-I-infected T-cell lines expressed high levels of MMP-9 compared with uninfected T-cell lines. In contrast, the levels of the related MMP-2 were not significantly altered by HTLV-I infection. In addition, the elevated expression of MMP-9 in HTLV-I-infected cells was attributable to the action of the viral transactivator protein Tax. The results show that Tax can activate the MMP-9 promoter and induce MMP-9 expression in T cells, indicating that the constitutive expression of MMP-9 in virus-infected cell lines is at least in part mediated by Tax. Activation of the MMP-9 promoter by Tax occurs mainly through the action of NF-kappaB and SP-1. The biologic significance of these observations was validated by the following 2 findings: MMP-9 expression was increased in primary ATL cells, and plasma MMP-9 levels were elevated in ATL patients. In addition, plasma levels of MMP-9 correlated with organ involvement in ATL patients. Together these data suggest that overexpression of MMP-9 in HTLV-I- infected cells may be in part responsible for the invasiveness of ATL cells.