RESUMEN
Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role.
Asunto(s)
Carbamatos/farmacología , Ácidos Carboxílicos/química , Inhibidores Enzimáticos/farmacología , Imidazoles/química , Insulisina/antagonistas & inhibidores , Insulisina/metabolismo , Oxadiazoles/farmacología , Carbamatos/síntesis química , Carbamatos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estructura Molecular , Oxadiazoles/síntesis química , Oxadiazoles/química , Relación Estructura-ActividadRESUMEN
Over the last decade, there has been a large effort to target aggrecanases, which are responsible for the degradation of the aggrecan in the extracellular matrix of joints, in order to hopefully lead to new treatments for osteoarthritis. Only a few inhibitors have been effective in explants or rodent models and thus only a few have reached the clinic, none of which have proven to be effective. In this article, a survey of chemical series is described, covering historical and recent inhibitors and highlighting how some of their problems were resolved, with a critical overview of the challenges encountered. A large effort should be undertaken in designing smaller compounds with higher residence times, defining new interaction sites on the aggrecanases and exploiting target flexibility.
Asunto(s)
Endopeptidasas/metabolismo , Osteoartritis/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Osteoartritis/enzimología , Osteoartritis/metabolismo , Inhibidores de Proteasas/química , Relación Estructura-ActividadRESUMEN
alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogeneous linear tridecapeptide with potential application for the modulation of skin tanning. To evaluate the interest of introducing a lipid moiety onto this peptide, we developed an efficient chemoselective parallel method to prepare a large series of analogues of alpha-melanocortin with high purity, varying the nature or the relative position of the lipid moiety. Two sets of building blocks containing lipidic alpha-oxo-aldehydes or alpha-hydrazinoacetyl peptides were combined to obtain a 102-membered library of amphiphilic alpha-MSH analogues. This library was pharmacologically tested at 1 x 10(-7) M for the ability to induce AMPc production in M4Be melanoma cell line after stimulation of the human melanocortin MC1 receptor. Among theses lipopeptides, 84 compounds exhibited an AMPc induction higher than Melitane, a patented alpha-MSH agonist. These results provide strong evidence of the interest of introduction of a lipid tail for the pharmacomodulation of bioactive peptides.
Asunto(s)
Hidrazonas/química , Lipoproteínas/síntesis química , Péptidos/síntesis química , alfa-MSH/química , Aldehídos/síntesis química , Secuencia de Aminoácidos , Línea Celular Tumoral/efectos de los fármacos , Cromatografía Liquida , Electroforesis , Humanos , Lipoproteínas/farmacología , Espectrometría de Masas , Melanoma/metabolismo , Melanoma/patología , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/farmacología , Receptor de Melanocortina Tipo 1/metabolismo , alfa-MSH/farmacologíaRESUMEN
BACKGROUND: Emergence of viral variants that escape CTL control is a major hurdle in HIV vaccination unless such variants affect gene regions that are essential for virus replication. Vaccine-induced multispecific CTL could also be able to control viral variants replication. To explore these possibilities, we extensively characterized CTL responses following vaccination with an epitope-based lipopeptide vaccine and challenge with pathogenic SIVmac251. The viral sequences corresponding to the epitopes present in the vaccine as well as the viral loads were then determined in every macaque following SIV inoculation. RESULTS: In most cases, the emergence of several viral variants or mutants within vaccine CTL epitopes after SIV challenge resulted in increased viral loads except for a single macaque, which showed a single escape viral variant within its 6 vaccine-induced CTL epitopes. CONCLUSION: These findings provide a better understanding of the evolution of CD8+ epitope variations after vaccination-induced CTL expansion and might provide new insight for the development of an effective HIV vaccine.
Asunto(s)
Productos del Gen nef/inmunología , Macaca mulatta/inmunología , Macaca mulatta/virología , Vacunas contra el SIDAS/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Animales , Productos del Gen nef/genética , Lipoproteínas/genética , Lipoproteínas/inmunología , Mutación/genética , ARN Viral/sangre , Vacunas contra el SIDAS/administración & dosificación , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Linfocitos T Citotóxicos/fisiología , Carga Viral , ViremiaRESUMEN
The selective delivery of antigens to professional antigen-presenting cells represents a promising approach to improve vaccine efficacy. Addition of a glycoamphiphile to a lipopeptide, whose interest for vaccination is now well-established, greatly favors its solubilization in aqueous solutions through the formation of mixed vesicles. Flow cytometry experiments indicate that this formulation does not diminish the uptake of the lipopeptide by the dendritic cells (DCs). These preliminary results suggest a possible straightforward, noncovalent targeting of cocktail-lipopeptide vaccines to the DCs via carbohydrate receptor-mediated endocytosis.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Glucosa/química , Glucosa/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Péptidos/metabolismo , Transporte Biológico , Células Cultivadas , Endocitosis , Citometría de Flujo , Humanos , Estructura Molecular , Péptidos/química , Rodaminas , Solubilidad , VacunasRESUMEN
Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation. Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayers of unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides were synthesized on the solid phase using N,N',N'-tri(tert-butyloxycarbonyl)-hydrazino acetic acid and further coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields were measured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology proved superior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields, implementation, and reproducibility. To check whether the grafted-peptides were accessible and functional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), which is involved in intracellular membrane trafficking, have been selected. Using this model, we demonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with the cytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting in cells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared by hydrazone chemoselective ligation, as a tool for biological investigation.
Asunto(s)
Hidrazonas/química , Liposomas/síntesis química , Sondas Moleculares/síntesis química , Péptidos/química , Complejo 3 de Proteína Adaptadora , Subunidades delta de Complexo de Proteína Adaptadora , Antígenos CD , Técnicas Químicas Combinatorias , Proteínas de Membrana de los Lisosomas , Péptidos/síntesis química , Transporte de Proteínas , Factores de Transcripción/metabolismoRESUMEN
Numerous clinical and experimental observations have shown that cellular immunity, in particular CD8+ T-lymphocytes, plays an important role in the control of HIV infection. We have focused on a lipopeptide vaccination strategy that has been shown to induce polyepitopic T-cell responses in both animals and humans, in order to deliver simian immunodeficiency virus (SIV) antigens to rhesus macaques. Given the relevance of antigen administration route in the development of an effective cellular immune response, this study was designed to assess SIV lipopeptide immunizations administered either by the intradermal (ID) or the intramuscular (IM) routes in their ability to elicit GAG and NEF multispecific T-lymphocytes in the rhesus macaque. Antigen specific T-cell responses were observed between 7 and 11 weeks following vaccination in both groups. Macaques immunized by the IM route yielded antigen-specific IFN-gamma secreting lymphocytes in response to no more than two pools of peptides derived from SIV-NEF. In contrast, among the four ID-immunized macaques, two presented multi-specific T-cell responses to as many as four pools of SIV-NEF and/or GAG peptides. Responses persisted 16 weeks following the vaccination protocol in one of the ID-vaccinated macaques. The induction of such responses is of great clinical relevance in the development of an effective HIV vaccine. Given the crucial role of CD8+ T-lymphocytes in HIV/SIV containment, vaccination through the intradermal route should merit high consideration in the development of an AIDS vaccine.
Asunto(s)
Vacunas contra el SIDAS/administración & dosificación , Virus de la Inmunodeficiencia de los Simios/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos/genética , Técnicas In Vitro , Inyecciones Intradérmicas , Lipoproteínas/administración & dosificación , Lipoproteínas/genética , Lipoproteínas/inmunología , Activación de Linfocitos , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Proteínas Virales/administración & dosificación , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Lipopeptides, a form of peptide immunogens, are currently under intense investigation as human vaccines for many infectious pathogens and cancers. However, the cellular and molecular mechanisms of lipopeptide immunogenicity are only partially understood. We have investigated the influence of the lipid content on the immunogenicity of lipopeptides using the herpes simplex virus type 1 (HSV-1) gD(1-23) peptide as a model antigen. Totally synthetic lipopeptides were constructed by covalent attachment to the peptide backbone of either Nepsilon-palmitoyl-lysine (palmitoyl-lipidated peptide, palmitoyl-LP) or cholesterol-lysine (cholesterol-lipidated peptide, cholesterol-LP). Immunization of mice with the palmitoyl-LP, but not with its cholesterol-LP analog, induced a strong T cell-dependent protective immunity against lethal HSV-1 infection. Analysis of cytokine profiles and IgG2a/IgG1 ratios revealed that a dominant Th1-type immune response was stimulated by the palmitoyl-LP, as opposed to a Th2 response generated by its cholesterol-LP analog. The palmitoyl-LP was efficiently taken up in vitro by immature dendritic cells (DC) in a time- and dose-dependent manner, and induced phenotypic maturation and production of pro-inflammatory cytokines by DC. Finally, DC stimulated with the palmitoyl-LP induced antigen-specific T cell responses through the Toll-like receptor-2 pathway. These findings have important implications for the development of effective lipopeptide immunization strategies against infectious pathogens.
Asunto(s)
Células Dendríticas/inmunología , Vacunas contra el Virus del Herpes Simple/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Lipoproteínas/inmunología , Glicoproteínas de Membrana/inmunología , Receptores de Superficie Celular/inmunología , Células TH1/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Colesterol/farmacología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/virología , Epítopos/inmunología , Femenino , Herpes Simple/prevención & control , Vacunas contra el Virus del Herpes Simple/farmacología , Lipoproteínas/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ácido Palmítico/farmacología , Receptor Toll-Like 2 , Receptores Toll-Like , Vacunación , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
We describe novel peptide-protein microarrays, which were fabricated using semicarbazide glass slides that permitted the immobilization of glyoxylyl peptides by site-specific ligation and the immobilization of proteins by physisorption. The arrays permitted the simultaneous serodetection of antibodies directed against hepatitis C virus (HCV core p21 15-45 peptide, NS4 1925-1947 peptide, core, NS3, NS4, and mixture of core, NS3, NS4, and NS5 antigens), hepatitis B virus (HBc, HBe, and HBs), human immunodeficiency virus (Gp41 and Gp120 for HIV-I and Gp36 for HIV-II), Epstein-Barr virus (VCAp18 153-176 peptide), and syphilis (rTpN47 and rTpN17) antigens using an immunofluorescence assay. Peptide-protein microarrays displayed high signal-to-noise ratios, sensitivities, and specificities for the detection of antibodies as revealed by the analysis of a collection of human sera referenced against these five pathogens.
Asunto(s)
Enfermedades Transmisibles/microbiología , Enfermedades Transmisibles/virología , Análisis por Matrices de Proteínas/métodos , Enfermedades Transmisibles/diagnóstico , Infecciones por Virus de Epstein-Barr/diagnóstico , Infecciones por Virus de Epstein-Barr/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Hepatitis/diagnóstico , Hepatitis/metabolismo , Hepatitis/virología , Virus de Hepatitis/genética , Virus de Hepatitis/aislamiento & purificación , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Péptidos/química , Péptidos/genética , Pruebas Serológicas/métodos , Treponema pallidum/química , Treponema pallidum/genética , Treponema pallidum/aislamiento & purificaciónRESUMEN
The ability of single-chain lipopeptides to gain access to cellular compartments other than those related to degradation/recycling was first deduced from their capacity to deliver peptide antigens into MHC-class I loading mechanisms. The ability of lipopeptides to escape complete endosome degradation was further illustrated by the selective inhibition of different protein kinase C isoenzymes and, more recently, the presentation of agonistic activity towards the interferon gamma receptor. Taken together, several independent results indicate that modification of a peptide by a single lipid chain confers upon it intracellular trafficking properties that can be used to deliver functional cargo peptides into living cells; the endoplasmic reticulum, cytosolic protease activity, sites of kinase activity, or even the signalling pathway associated with cytokine stimulation, all appear accessible to peptide modified by a single lipidic moiety. In this context, the interferon gamma receptor can be considered as a very discriminative pharmacological model, useful for the comparative evaluation of the cellular delivery of lipopeptides, as it allows the unambiguous tracking of their intact delivery into a wide range of cellular compartments. This model is now being used to probe the influence of the nature of the lipid moiety on the trafficking properties of lipopeptides.
Asunto(s)
Sistemas de Liberación de Medicamentos , Lípidos , Péptidos/administración & dosificación , Animales , Transporte Biológico Activo/fisiología , Membrana Celular/metabolismo , Sistemas de Liberación de Medicamentos/tendencias , Predicción , Humanos , Interferón gamma/metabolismo , Péptidos/farmacocinéticaRESUMEN
We studied the effect of booster injections and the long-term immune response after injections of an anti-human immunodeficiency virus type 1 (HIV-1) lipopeptide vaccine. This vaccine was injected alone or with QS21 adjuvant to 28 HIV-uninfected volunteers. One month later, after a fourth injection of the vaccine, B- and T-cell anti-HIV responses were detected in >85% of the vaccinated volunteers. One year after this injection, a long-term immune response was observed in >50% of the volunteers. At this point, a positive QS21 effect was observed only in the sustained B-cell and CD4(+)-T-cell responses. To better characterize the CD8(+)-T-cell response, we used a gamma interferon enzyme-linked immunospot method and a bank of 59 HIV-1 epitopes. For the six most common HLA molecules (HLA-A2, -A3, -A11, -A24, -B7 superfamily, and -B8), an average of 10 (range, 3 to 15) HIV-1 epitopes were tested. CD8(+)-T-cell responses were evaluated according to the HLA class I molecules of the volunteers. Each assessment was based on 18 HIV-1 epitopes in average. We showed that 31 HIV-1 epitopes elicited specific CD8(+)-T-cell responses after vaccination. The most frequently recognized peptides were Nef 68-76 (-B7), Nef 71-79 (-B7), Nef 84-92 (-A11), Nef 135-143 (-B7), Nef 136-145 (-A2), Nef 137-145 (-A2), Gag 259-267 (-B8), Gag 260-268 (-A2), Gag 267-274 (-A2), Gag 267-277 (-B7), and Gag 276-283 (A24). We found that CD8(+)-T-cell epitopes were induced at a higher number after a fourth injection (P < 0.05 compared to three injections), which indicates an increase in the breadth of HIV CD8(+)-T-cell epitope recognition after the boost.
Asunto(s)
Vacunas contra el SIDA/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T , VIH-1/inmunología , Lipoproteínas/inmunología , Humanos , Inmunización Secundaria , Activación de LinfocitosRESUMEN
The molecular characterization of the epitope repertoire on herpes simplex virus (HSV) antigens would greatly expand our knowledge of HSV immunity and improve immune interventions against herpesvirus infections. HSV glycoprotein D (gD) is an immunodominant viral coat protein and is considered an excellent vaccine candidate antigen. By using the TEPITOPE prediction algorithm, we have identified and characterized a total of 12 regions within the HSV type 1 (HSV-1) gD bearing potential CD4(+) T-cell epitopes, each 27 to 34 amino acids in length. Immunogenicity studies of the corresponding medium-sized peptides confirmed all previously known gD epitopes and additionally revealed four new immunodominant regions (gD(49-82), gD(146-179), gD(228-257), and gD(332-358)), each containing naturally processed epitopes. These epitopes elicited potent T-cell responses in mice of diverse major histocompatibility complex backgrounds. Each of the four new immunodominant peptide epitopes generated strong CD4(+) Th1 T cells that were biologically active against HSV-1-infected bone marrow-derived dendritic cells. Importantly, immunization of H-2(d) mice with the four newly identified CD4(+) Th1 peptide epitopes but not with four CD4(+) Th2 peptide epitopes induced a robust protective immunity against lethal ocular HSV-1 challenge. These peptide epitopes may prove to be important components of an effective immunoprophylactic strategy against herpes.
Asunto(s)
Antígenos Virales/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/inmunología , Epítopos Inmunodominantes/genética , Células TH1/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Algoritmos , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Herpes Simple/inmunología , Herpes Simple/prevención & control , Vacunas contra Herpesvirus/genética , Vacunas contra Herpesvirus/farmacología , Inmunización , Epítopos Inmunodominantes/química , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia MolecularRESUMEN
A novel procedure for the preparation of alpha-hydrazinoacetyl peptides is reported on the basis of the solid-phase coupling of partially or fully Boc-protected hydrazino acetic acid derivatives. The degree of unwanted polymerization of the activated ester during both activation and coupling was found to be significant for the monoprotected derivative BocNHNHCH(2)CO(2)H but could be minimized with the diprotected derivative BocNHNH(Boc)CH(2)CO(2)H and suppressed with the fully protected acid. Despite the instability of the imidocarbonate group toward acids and bases, a low-cost and effective route was sought for the preparation of the tris(Boc)-protected derivative. The N,N,N'-tris(Boc)hydrazinoacetic acid could be introduced on the solid phase after or before peptide elongation using Fmoc/tert-butyl chemistry. In this latter case, HR MAS NMR analysis of model solid supports demonstrated the partial loss of one Boc group during the repetitive piperidine treatments. Despite this slight instability, N,N,N'-tris(Boc)hydrazinoacetic acid was found to be a highly convenient reagent for the robust and easily scalable preparation of hydrazinopeptides in good yield and high purity.
Asunto(s)
Hidrazinas/química , Péptidos/química , Secuencia de Aminoácidos , Hidrazinas/síntesis química , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Péptidos/síntesis químicaRESUMEN
We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Péptidos/química , Semicarbacidas/química , Aldehídos/química , Anticuerpos/química , Biotina/química , Biotina/inmunología , Tampones (Química) , Colorantes Fluorescentes , Hidrólisis , Hibridación in Situ , Indicadores y Reactivos , Oligonucleótidos/genética , Control de Calidad , Silanos/química , Estreptavidina/químicaRESUMEN
The modification of a peptide antigen by a fatty acid such as palmitic acid is now recognized as a mean to induce cellular responses. Mixtures of lipopeptides, obtained by combining individually synthesized compounds, were shown to be promising synthetic vaccine candidates. Usually, in lipopeptide synthesis, the fatty acyl moiety is introduced on the crude peptide chain using solid-phase methods. The separation of the target compound from impurities by RP-HPLC is often complicated by the amphiphilic properties of lipopeptides and results in low overall yields. To overcome the difficulties associated with lipopeptide synthesis and mixture preparation, we have developed a method where the fatty acyl moiety is site-specifically and collectively introduced in solution onto a mixture of individually prepurified peptides. The lipidation is based on the quasistoichiometric and high-yielding ligation of a glyoxylyl lipid with hydrazinoacetyl peptides. The hydrazone constructs were prepared in a salt-free medium and could be isolated by direct lyophilization of the reaction mixture. This process is compatible with cysteinyl peptides, and no aggregation nor degradation could be observed.
Asunto(s)
Lípidos/química , Péptidos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cisteína/química , Hidrazonas/química , Indicadores y Reactivos , Ligandos , Espectrometría de Masas , Datos de Secuencia Molecular , Virus de la Inmunodeficiencia de los Simios/químicaRESUMEN
A practical preparation of onion vesicles targeted to dendritic cells involves the grafting of mannose-mimetic clusters, bearing a hydrazino group, onto the surface of onion vesicles containing an aldehyde functionalized lipid.
Asunto(s)
Células Dendríticas/metabolismo , Lectinas Tipo C/metabolismo , Liposomas/farmacocinética , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Técnicas Químicas Combinatorias , Células Dendríticas/química , Sistemas de Liberación de Medicamentos/métodos , Humanos , Ligandos , Liposomas/síntesis química , Liposomas/uso terapéutico , Receptor de Manosa , Microscopía ConfocalRESUMEN
Effect of charge and shape of multivalent lysine-based cluster glycomimetics on their mannose receptor-mediated uptake by human dendritic cells has been evaluated: The capture is strongly affected by the shape of the ligands. The effect of charge is less pronounced although positive charges on the ligands seem to favor non-specific endocytosis capture.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Glucosa/farmacología , Lectinas Tipo C , Lectinas de Unión a Manosa , Receptores de Superficie Celular/efectos de los fármacos , Citometría de Flujo , Fluoresceína , Ligandos , Receptor de Manosa , Imitación Molecular , Ácido Quínico/química , Ácido Shikímico/farmacologíaRESUMEN
We recently reported that parenteral injection of malaria palmitoyl-tailed peptides without adjuvant efficiently induces B, Th and CTL responses. We now show that intranasal (IN) or sub-lingual (SL) delivery of such lipopeptides induces strong systemic immune responses, as demonstrated by specific Th cell responses from the spleen as well as inguinal lymph nodes, and by the production ofhigh levels of serum antibodies. Overall, both types of responses were significantly higher than in parallel experiments in which the same lipopeptides were delivered by the subcutaneous (s.c.) route. Moreover, the mucosal route resulted in the preferential induction of IFN-gamma producing T cells and of IgG2a antibody production, as compared to the dominant IL-4 and IgG1 responses obtained by the s.c. route, thus bringing a distinct advantage in the field of many infectious diseases and allergy. Possibly related to this Th1 response, we found that dendritic cells, the principal immune-competent cells to encounter antigens within mucosal membranes, take up lipopeptide antigens more efficiently than macrophages. Mucosal immunization by lipidated peptides appears therefore as a novel, noninvasive vaccine approach that does not require the use of extraneous adjuvant and which, besides cost-effectiveness, has attractive practical and immunological features.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos de Protozoos/inmunología , Linfocitos T/inmunología , Administración Intranasal , Administración Sublingual , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/administración & dosificación , Células Dendríticas/fisiología , Inmunización , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Células TH1/inmunologíaRESUMEN
We investigated the relationships between class II human leukocyte antigens (HLA) and the antibody response to Plasmodium falciparum p126 protein and to its amino-terminal portion (Nt47) in 2 malaria-endemic villages in Brazil, Colina and Ribeirinha. All people from the endemic areas had anti-p126 antibodies, and the frequencies of anti-Nt47 antibodies were similar in both communities (66% for Colina and 75% for Ribeirinha). Typing of HLA showed that Colina and Ribeirinha groups had no significant differences in HLA antigen frequencies. However, in both groups, significant associations between positive response to anti-Nt47 and presence of HLA-DR4, as well as between absence of response and presence of HLA-DR15, were observed. The predominance of positive responses to Nt47 among HLA-DR4 people was independent of the presence of any particular allele. There was no evidence for association between HLA-DQB1 alleles and antibody response to Nt47. Thus, naturally exposed people with different HLA class II antigens seem to respond differently to Nt47, indicating that the choice of relevant peptide sequences may have important consequences for subunit vaccine development.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Antígenos HLA-D/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adulto , Alelos , Animales , Formación de Anticuerpos , Brasil , Femenino , Genes MHC Clase II , Geografía , Antígenos HLA-DR/análisis , Subtipos Serológicos HLA-DR , Antígeno HLA-DR4/análisis , Prueba de Histocompatibilidad , Humanos , Malaria Falciparum/inmunología , MasculinoRESUMEN
We report a novel generation of peptide arrays fabricated by site-specific ligation of glyoxylyl peptides onto glass slides covered by a semicarbazide sol-gel layer. These arrays allowed the highly sensitive and specific detection of antibodies in very small blood samples from infected individuals using three model peptidic epitopes (HCV Core and NS4, EBV Capsid) in an immunofluorescence assay. Comparison with standard enzyme-linked immunosorbent assays (ELISAs) demonstrated a large gain in sensitivity and specificity. These unique properties, combined with the possibility to immobilize glycoproteins such as antibodies, offer the possibility to perform sandwich immunofluorescent assays in a highly parallel format.