RESUMO
Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4+ T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P < 0.001) response kinetics and superior ADCC (P < 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.
Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Antígenos CD4/imunologia , Anticorpos Anti-HIV/imunologia , Macaca mulatta/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , HIV-1/imunologia , Macaca mulatta/virologia , VacinaçãoRESUMO
CD4 binding on gp120 leads to the exposure of highly conserved regions recognized by the HIV co-receptor CCR5 and by CD4-induced (CD4i) antibodies. A covalent gp120-CD4 complex was shown to elicit CD4i antibody responses in monkeys, which was correlated with control of the HIV virus infection (DeVico, A., Fouts, T., Lewis, G. K., Gallo, R. C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 17477-17482). Because the inclusion of CD4 in a vaccine formulation should be avoided, due to potential autoimmune reactions, we engineered small sized CD4 mimetics (miniCD4s) that are poorly immunogenic and do not induce anti-CD4 antibodies. We made covalent complexes between such an engineered miniCD4 and gp120 or gp140, through a site-directed coupling reaction. These complexes were recognized by CD4i antibodies as well as by the HIV co-receptor CCR5. In addition, they elicit CD4i antibody responses in rabbits and therefore represent potential vaccine candidates that mimic an important HIV fusion intermediate, without autoimmune hazard.
Assuntos
Linfócitos T CD4-Positivos/virologia , Proteína gp120 do Envelope de HIV/química , HIV-1/química , Proteínas do Envelope Viral/química , Animais , Apresentação de Antígeno , Células CHO , Cricetinae , Cricetulus , Reagentes de Ligações Cruzadas/química , Cisteína/química , Dissulfetos , Ligação Proteica , Conformação Proteica , Receptores CCR5/químicaRESUMO
Replicative vectors derived from live-attenuated measles virus (MV) carrying additional non-measles vaccine antigens have long demonstrated safety and immunogenicity in humans despite pre-existing immunity to measles. Here, we report the vaccination of cynomolgus macaques with MV replicative vectors expressing simian-human immunodeficiency virus Gag, Env, and Nef antigens (MV-SHIV Wt) either wild type or mutated in the immunosuppressive (IS) domains of Nef and Env antigens (MV-SHIV Mt). We found that the inactivation of Nef and Env IS domains by targeted mutations led to the induction of significantly enhanced post-prime cellular immune responses. After repeated challenges with low doses of SHIV-SF162p3, vaccinees were protected against high viremia, resulting in a 2-Log reduction in peak viremia, accelerated viral clearance, and a decrease -even complete protection for nearly half of the monkeys- in reservoir cell infection. This study demonstrates the potential of a replicative viral vector derived from the safe and widely used measles vaccine in the development of a future human vaccine against HIV-1.
RESUMO
Purpose: To determine the plasma metabolomic signature of primary open-angle glaucoma (POAG). Methods: We compared the metabolomic profiles of plasma from individuals with POAG (n = 36) with age- and sex-matched controls with cataract (n = 27). A targeted metabolomics study was performed using the standardized p180 Biocrates Absolute IDQ p180 kit with a QTRAP 5500 mass spectrometer. Multivariate analyses were performed using principal component analysis (PCA) and the least absolute shrinkage and selection operator (LASSO) method. Results: Among the 151 metabolites accurately measured, combined univariate and multivariate analyses revealed 18 discriminant metabolites belonging to the carbohydrate, acyl-carnitine, phosphatidylcholine, amino acids, and polyamine families. The metabolomic signature of POAG points to three closely interdependent pathophysiologic conditions; that is, defective mitochondrial oxidation of energetic substrates, altered metabolism resembling that observed in senescence, and a deficiency in spermidine and spermine, both polyamines being involved in the protection of retinal ganglion cells. Conclusions: Our results highlight a systemic and age-related mitochondrial defect in the pathogenesis of POAG.
Assuntos
Envelhecimento , Proteínas do Olho/sangue , Glaucoma de Ângulo Aberto/sangue , Metaboloma , Metabolômica/métodos , Doenças Mitocondriais/sangue , Espermidina/sangue , Espermina/sangue , Idoso , Feminino , Humanos , Masculino , Espectrometria de Massas , Análise de Componente PrincipalRESUMO
Purpose: To determine the plasma metabolomic signature of the exfoliative syndrome (XFS), the most common cause worldwide of secondary open-angle glaucoma. Methods: We performed a targeted metabolomic study, using the standardized p180 Biocrates Absolute IDQ p180 kit with a QTRAP 5500 mass spectrometer, to compare the metabolomic profiles of plasma from individuals with XFS (n = 16), and an age- and sex-matched control group with cataract (n = 18). Results: A total of 151 metabolites were detected correctly, 16 of which allowed for construction of an OPLS-DA model with a good predictive capability (Q2cum = 0.51) associated with a low risk of over-fitting (permQ2 = -0.48, CV-ANOVA P-value <0.001). The metabolites contributing the most to the signature were octanoyl-carnitine (C8) and decanoyl-carnitine (C10), the branched-chain amino acids (i.e., isoleucine, leucine, and valine), and tyrosine, all of which were at higher concentrations in the XFS group, whereas spermine and spermidine, together with their precursor acetyl-ornithine, were at lower concentrations than in the control group. Conclusions: We identified a significant metabolomic signature in the plasma of individuals with XFS. Paradoxically, this signature, characterized by lower concentrations of the neuroprotective spermine and spermidine polyamines than in controls, partially overlaps the plasma metabolomic profile associated with insulin resistance, despite the absence of evidence of insulin resistance in XFS.
Assuntos
Aminoácidos/sangue , Biomarcadores/sangue , Carnitina/análogos & derivados , Síndrome de Exfoliação/sangue , Glaucoma de Ângulo Aberto/sangue , Metaboloma/fisiologia , Poliaminas/sangue , Idoso , Carnitina/sangue , Feminino , Humanos , Masculino , Espectrometria de Massas , Metabolômica/métodosRESUMO
In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/mL) by an increased DNA binding activity of NF-kappaB and AP-1 transcription factors. Incubation of the cells with 10(-5) M rhein for 24 h was found to reduce this activity, particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to 1-h treatment with IL-1beta. This effect was greater in hypoxia (3% O2) than in normoxia (21% O2). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The level of c-jun protein, an element of AP-1 complex, was increased by exposure of the chondrocytes to IL-1beta after 2, 6, and 24 h. Addition of rhein at 10(-5) M for 24 h did not reduce the c-jun protein amount. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extracellular matrix synthesis. IL-1-induced collagenase (MMPI) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several proinflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMPI synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the antiosteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.
Assuntos
Antraquinonas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Bovinos , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Interleucina-1/antagonistas & inibidores , Interleucina-1/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , NF-kappa B/antagonistas & inibidores , Osteoartrite/prevenção & controle , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Fator de Transcrição AP-1/antagonistas & inibidoresRESUMO
The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M) and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application.
Assuntos
Anticorpos Neutralizantes/metabolismo , Formação de Anticorpos , Antígenos CD4/imunologia , HIV-1/imunologia , Proteínas Recombinantes/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/síntese química , Vacinas contra a AIDS/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Biomimética , Reagentes de Ligações Cruzadas/farmacologia , Epitopos/imunologia , Feminino , Anticorpos Anti-HIV/metabolismo , Imunização , Testes de Neutralização , Coelhos , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/farmacologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
To counteract the problems associated with the purification of HIV envelope, we developed a new purification method exploiting the high affinity of a peptide mimicking CD4 towards the viral glycoprotein. This miniCD4 was used as a ligand in affinity chromatography and allowed the separation in one step of HIV envelope monomer from cell supernatant and the capture of pre-purified trimer. This simple and robust method of purification yielded to active and intact HIV envelopes as proved by the binding of CCR5 HIV co-receptor, CD4 and a panel of well-characterized monoclonal antibodies. The immunogenicity of miniCD4-purified HIV envelope was further assessed in rats. The analysis of the humoral response indicated that elicited antibodies were able to recognize a broad range of HIV envelopes. Finally, this method based on a chemically synthesized peptide may represent a convenient and versatile tool for protein purification compatible far scale-up in both academic and pharmaceutical researches.
Assuntos
Antígenos CD4/metabolismo , Cromatografia de Afinidade/métodos , Proteína gp120 do Envelope de HIV/isolamento & purificação , HIV-1 , Receptores CCR5/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/imunologia , Peptídeos/química , Ligação Proteica , Multimerização Proteica , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismoRESUMO
Interleukin-1 beta (IL-1 beta) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1 beta inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1 beta down-regulates COL2A1 gene transcription through a -41/-33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1 beta effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type -50/+1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1 beta inhibition of a -63/+47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the -41/-33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the -63/+1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1 beta down-regulation of that gene, as we found previously for transforming growth factor-beta 1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1 beta increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1 beta decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.