RESUMO
Human immunodeficiency virus (HIV)-1 infection of the central nervous system occurs in the vast majority of HIV-infected patients. HIV-associated dementia (HAD) represents the most severe form of HIV-related neuropsychiatric impairment and is associated with neuropathology involving HIV proteins and activation of proinflammatory cytokine circuits. Interferon-gamma (IFN-gamma) activates the JAK/STAT1 pathway, a key regulator of inflammatory and apoptotic signaling, and is elevated in HIV-1-infected brains progressing to HAD. Recent reports suggest green tea-derived (-)-epigallocatechin-3-gallate (EGCG) can attenuate neuronal damage mediated by this pathway in conditions such as brain ischemia. In order to investigate the therapeutic potential of EGCG to mitigate the neuronal damage characteristic of HAD, IFN-gamma was evaluated for its ability to enhance well-known neurotoxic properties of HIV-1 proteins gp120 and Tat in primary neurons and mice. Indeed, IFN-gamma enhanced the neurotoxicity of gp120 and Tat via increased JAK/STAT signaling. Additionally, primary neurons pretreated with a JAK1 inhibitor, or those derived from STAT1-deficient mice, were largely resistant to the IFN-gamma-enhanced neurotoxicity of gp120 and Tat. Moreover, EGCG treatment of primary neurons from normal mice reduced IFN-gamma-enhanced neurotoxicity of gp120 and Tat by inhibiting JAK/STAT1 pathway activation. EGCG was also found to mitigate the neurotoxic properties of HIV-1 proteins in the presence of IFN-gamma in vivo. Taken together, these data suggest EGCG attenuates the neurotoxicity of IFN-gamma augmented neuronal damage from HIV-1 proteins gp120 and Tat both in vitro and in vivo. Thus EGCG may represent a novel natural copound for the prevention and treatment of HAD.
Assuntos
Complexo AIDS Demência/tratamento farmacológico , Catequina/análogos & derivados , HIV-1/imunologia , Interferon gama/metabolismo , Janus Quinases/metabolismo , Neurônios/enzimologia , Fármacos Neuroprotetores/uso terapêutico , Fator de Transcrição STAT1/metabolismo , Complexo AIDS Demência/enzimologia , Complexo AIDS Demência/imunologia , Complexo AIDS Demência/patologia , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Catequina/uso terapêutico , Células Cultivadas , Feminino , Produtos do Gene tat/imunologia , Produtos do Gene tat/toxicidade , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/toxicidade , Interferon gama/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/imunologia , Neurônios/patologia , Neurotoxinas/imunologia , Neurotoxinas/toxicidade , Fator de Transcrição STAT1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The generation of a vaccine against HIV/AIDS is extremely challenging, as evidenced by more than 20 years of attempts. Here are highlighted the strategies adopted within the AIDS Vaccine Integrated project (AVIP) to speed up the clinical evaluation of novel vaccine candidates and to increase the chances to get an effective preventive and/or therapeutic vaccine.
Assuntos
Vacinas contra a AIDS , HIV-1/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Animais , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene tat/genética , Produtos do Gene tat/imunologia , Genes Virais , Antígenos HIV/genética , Antígenos HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/genética , Humanos , Modelos Animais , Vacinas Combinadas/genética , Vacinas Combinadas/imunologia , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
HIV-Tat, a conserved protein playing a key role in the early life cycle of the human immunodeficiency virus (HIV) has been proposed as a potential AIDS vaccine. An HIV-Tat-based vaccine should elicit a broad, long-lasting, and neutralizing immune response. We have previously demonstrated that the adenylate cyclase (CyaA) from Bordetella pertussis targets dendritic cells and delivers CD8(+) and CD4(+) T-cell epitopes into the major histocompatibility complex class I and class II presentation pathways. We have also showed that CyaA induced specific and protective cytotoxic T cell responses in vivo. Here, we designed a prototype vaccine based on the HIV type 1 Tat delivered by CyaA (CyaA-E5-Tat) and tested its capacity to induce HIV-Tat-specific cellular as well as antibody responses. We showed that immunization of mice by CyaA-E5-Tat in the absence of adjuvant elicited strong and long-lasting neutralizing anti-Tat antibody responses more efficient than those obtained after immunization with Tat toxoid in aluminum hydroxide adjuvant. Analyses of the anti-Tat immunoglobulin G isotypes and the cytokine pattern showed that CyaA-E5-Tat induced a Th1-polarized immune response in contrast to the Th2-polarized immune responses obtained with the Tat toxoid. In addition, our data demonstrated that HIV-Tat-specific gamma interferon-producing CD8(+) T cells were generated after vaccination with CyaA-E5-Tat in a CD4(+) T-cell-independent manner. Based on these findings, CyaA-E5-Tat represents an attractive vaccine candidate for both preventive and therapeutic vaccination involving CyaA as an efficient nonreplicative vector for protein delivery.
Assuntos
Vacinas contra a AIDS/imunologia , Toxina Adenilato Ciclase/imunologia , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene tat/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Imunização , Toxina Adenilato Ciclase/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Infecções por HIV/sangue , Interferon gama/biossíntese , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Baço/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Células Th1/imunologia , Vacinas Sintéticas/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The immunotherapeutic potential of biologically active HIV-1 Tat protein coupled to autologous red blood cells (RBCs) was evaluated in a mouse model. HIV-1 Tat expressed in Escherichia coli and purified to homogeneity was found to be active in viral trans activation and efficiently internalised by monocyte-derived dendritic cells (MDDCs). The product of HIV-Tat biotinylation and coupling to RBCs by means of a biotin-avidin-biotin bridge, (RBC-Tat), showed no trans activation activity and was still efficiently internalized by MDDCs as compared to uncoupled Tat.Balb/c mice were then immunized with 10 microg of soluble Tat in complete Freund's adjuvant or with 40 ng of Tat coupled on RBCs surface and boosted at week 3, 6 and 25 with 5 microg soluble Tat in incomplete Freund's adjuvant or with 20 ng of RBC-coupled Tat, respectively. Anti-Tat antibody response was similar in both groups; however, 2/6 animals immunized with soluble Tat and 6/6 animals immunized with RBC-Tat developed anti-Tat neutralizing antibodies. In addition, at week 28 cytolytic anti-Tat CTLs were detected in all animals although they were slightly higher in mice immunized with RBC-Tat. These results indicate that RBC-mediated delivery of HIV-1 Tat, in amounts 250 times lower than soluble Tat, is safe and induces specific CTL responses and neutralizing antibodies.
Assuntos
Vacinas contra a AIDS/imunologia , Transfusão de Eritrócitos , Produtos do Gene tat/genética , HIV-1/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Biotinilação , Produtos do Gene tat/imunologia , Esquemas de Imunização , Camundongos , Proteínas Recombinantes/imunologia , Transplante Autólogo , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Although Tat-specific CTL responses are elicited in HIV-infected patients and in non-human primate models, specific CTL epitopes within Tat have not been identified. In this study, we mucosally immunized mice with recombinant, full-length Tat protein or individual Tat-specific, overlapping peptides to map putative H-2d-restricted, Tat-specific CTL epitopes. Standard chromium release assays from splenocytes of immunized animals identified a peptide (QPKTACTNC) capable of inducing Tat-specific CTL responses. This newly-identified epitope lies within a region of low sequence variability among HIV-1 subtypes, suggesting its potential use in a multicomponent AIDS vaccine.
Assuntos
Vacinas contra a AIDS/imunologia , Proteínas de Escherichia coli , Produtos do Gene tat/imunologia , Antígenos HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas contra a AIDS/administração & dosagem , Adjuvantes Imunológicos , Administração Intranasal , Sequência de Aminoácidos , Animais , Toxinas Bacterianas/imunologia , Testes Imunológicos de Citotoxicidade , Enterotoxinas/imunologia , Epitopos/imunologia , Produtos do Gene tat/química , Antígenos HIV/química , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Proteínas Recombinantes/imunologia , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
We have here investigated the effect of the regulatory Tat protein of the human immunodeficiency virus type 1 (HIV-1) on the PI 3-kinase catalytic activity in PC12 rat pheochromocytoma cells. After as early as 1 min from the beginning of the treatment with recombinant HIV-1 Tat protein, a significant increase in the tyrosine phosphorylation levels of the p85 regulatory subunit of PI 3-kinase was noticed in 48 h serum-starved PC12 cells. Moreover, the addition of Tat to PC12 cells induced a great increase in PI 3-kinase immunoprecipitated with an anti-phosphotyrosine antibody with a peak of activity (19-fold increase with respect to the basal levels) after a 15-min treatment. This increase in PI 3-kinase activity was significantly higher in PC12 cell cultures supplemented with Tat protein than in cultures stimulated by 100 ng/ml nerve growth factor (NGF; 8-fold increase with respect to the basal levels). Further experiments showed that Tat protein was able to specifically activate PI 3-kinase at picomolar concentrations. In fact: (i) maximal activation of PI 3-kinase was observed at concentrations as low as 1 ng/ml and was specifically blocked by anti-Tat neutralizing antibody; (ii) a Tat-dependent activation was also observed in experiments in which PI 3-kinase activity was evaluated in either anti-Tyr(P) or anti-p85 immunoprecipitates; (iii) 100 nM wortmannin completely blocked the Tat-mediated increase in PI 3-kinase activity both in vitro and in vivo. Our data strongly support the concept that extracellular Tat acts as a cell stimulator, inducing intracellular signal transduction in uninfected cells.