RESUMEN

BACKGROUND: Soluble oligomeric (misfolded) species of amyloid-ß (Aß) are the main mediators of toxicity in Alzheimer's disease (AD). These oligomers subsequently form aggregates of insoluble fibrils that precipitate as extracellular and perivascular plaques in the brain. Active immunization against Aß is a promising disease modifying strategy. However, eliciting an immune response against Aß in general may interfere with its biological function and was shown to cause unwanted side-effects. Therefore, we have developed a novel experimental vaccine based on conformational neo-epitopes that are exposed in the misfolded oligomeric Aß, inducing a specific antibody response. OBJECTIVE: Here we investigate the protective effects of the experimental vaccine against oligomeric Aß1-42-induced neuronal fiber loss in vivo. METHODS: C57BL/6 mice were immunized or mock-immunized. Antibody responses were measured by enzyme-linked immunosorbent assay. Next, mice received a stereotactic injection of oligomeric Aß1-42 into the nucleus basalis of Meynert (NBM) on one side of the brain (lesion side), and scrambled Aß1-42 peptide in the contralateral NBM (control side). The densities of choline acetyltransferase-stained cholinergic fibers origination from the NBM were measured in the parietal neocortex postmortem. The percentage of fiber loss in the lesion side was determined relative to the control side of the brain. RESULTS: Immunized responders (79%) showed 23% less cholinergic fiber loss (p = 0.01) relative to mock-immunized mice. Moreover, fiber loss in immunized responders correlated negatively with the measured antibody responses (R2 = 0.29, p = 0.02). CONCLUSION: These results may provide a lead towards a (prophylactic) vaccine to prevent or at least attenuate (early onset) AD symptoms.

Asunto(s)

Péptidos beta-Amiloides/química , Inmunización/métodos , Enfermedades Neurodegenerativas , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Péptidos Cíclicos/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/toxicidad , Animales , Núcleo Basal de Meynert/metabolismo , Núcleo Basal de Meynert/patología , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/terapia , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad

RESUMEN

The 39- to 42-residue amyloid ß (Aß) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aß immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aß, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aß-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aß(22-28)-YNGK' elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound--although more weakly--to Aß(1-42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aß deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.

Asunto(s)

Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/inmunología , Anticuerpos/inmunología , Péptidos Cíclicos/química , Péptidos Cíclicos/inmunología , Placa Amiloide/inmunología , Secuencia de Aminoácidos , Animales , Antígenos/inmunología , Western Blotting , Encéfalo/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína

RESUMEN

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.

Asunto(s)

Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígeno HLA-DR1/metabolismo , Vacunas Meningococicas/inmunología , Neisseria meningitidis/inmunología , Fragmentos de Péptidos/inmunología , Porinas/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/metabolismo , Linfocitos T CD4-Positivos/microbiología , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígeno HLA-DR1/biosíntesis , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/inmunología , Homocigoto , Humanos , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Vacunas Meningococicas/genética , Vacunas Meningococicas/metabolismo , Datos de Secuencia Molecular , Neisseria meningitidis/aislamiento & purificación , Neisseria meningitidis/metabolismo , Nitrógeno/metabolismo , Isótopos de Nitrógeno/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Porinas/genética , Porinas/metabolismo

RESUMEN

Formaldehyde is a well known cross-linking agent that can inactivate, stabilize, or immobilize proteins. The purpose of this study was to map the chemical modifications occurring on each natural amino acid residue caused by formaldehyde. Therefore, model peptides were treated with excess formaldehyde, and the reaction products were analyzed by liquid chromatography-mass spectrometry. Formaldehyde was shown to react with the amino group of the N-terminal amino acid residue and the side-chains of arginine, cysteine, histidine, and lysine residues. Depending on the peptide sequence, methylol groups, Schiff-bases, and methylene bridges were formed. To study intermolecular cross-linking in more detail, cyanoborohydride or glycine was added to the reaction solution. The use of cyanoborohydride could easily distinguish between peptides containing a Schiff-base or a methylene bridge. Formaldehyde and glycine formed a Schiff-base adduct, which was rapidly attached to primary N-terminal amino groups, arginine and tyrosine residues, and, to a lesser degree, asparagine, glutamine, histidine, and tryptophan residues. Unexpected modifications were found in peptides containing a free N-terminal amino group or an arginine residue. Formaldehyde-glycine adducts reacted with the N terminus by means of two steps: the N terminus formed an imidazolidinone, and then the glycine was attached via a methylene bridge. Two covalent modifications occurred on an arginine-containing peptide: (i) the attachment of one glycine molecule to the arginine residue via two methylene bridges, and (ii) the coupling of two glycine molecules via four methylene bridges. Remarkably, formaldehyde did not generate intermolecular cross-links between two primary amino groups. In conclusion, the use of model peptides enabled us to determine the reactivity of each particular cross-link reaction as a function of the reaction conditions and to identify new reaction products after incubation with formaldehyde.

Asunto(s)

Formaldehído/farmacología , Proteínas/química , Cromatografía Liquida , Reactivos de Enlaces Cruzados/farmacología , Formaldehído/química , Glicina/química , Lisina/química , Espectrometría de Masas , Modelos Químicos , Péptidos/química , Estructura Terciaria de Proteína , Serina Endopeptidasas/química , Factores de Tiempo

RESUMEN

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.

Asunto(s)

Vacunas de Subunidad/química , Vacunas de Subunidad/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Vacunas Bacterianas/química , Vacunas Bacterianas/inmunología , Reacciones Cruzadas , Diseño de Fármacos , Inmunización , Técnicas In Vitro , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/inmunología , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Péptidos Cíclicos/inmunología , Porinas/química , Porinas/genética , Porinas/inmunología , Conformación Proteica , Ingeniería de Proteínas , Termodinámica , Vacunas de Subunidad/genética

RESUMEN

Memory CD4 T-cell responses against respiratory syncytial virus (RSV) were evaluated in peripheral blood mononuclear cells of healthy blood donors with gamma interferon enzyme-linked immunospot (Elispot) assays. RSV-specific responses were detected in every donor at levels varying between 0.05 and 0.3% of CD4 T cells. For all donors tested, a considerable component of the CD4 T-cell response was directed against the fusion (F) protein of RSV. We characterized a set of 31 immunodominant antigenic peptides targeted by CD4 T cells in the context of the most prevalent HLA class II molecules within the Caucasian population. Most antigenic peptides were HLA-DR restricted, whereas two dominant DQ peptides were also identified. The antigenic peptides identified were located across the entire sequence of the F protein. Several peptides were presented by more than one major histocompatibility complex class II molecule. Furthermore, most donors recognized several F peptides. Detailed knowledge about immunodominant antigenic peptides will facilitate the ability to monitor CD4 T-cell responses in patients and the measurement of correlates of protection in vaccinated subjects.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Epítopos Inmunodominantes/análisis , Proteínas Virales de Fusión/inmunología , Proteínas Virales/inmunología , Adulto , Secuencia de Aminoácidos , Ensayo de Inmunoadsorción Enzimática , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/inmunología , Datos de Secuencia Molecular