RESUMEN
BACKGROUND: The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity. METHODS: Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders. RESULTS: For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes. CONCLUSIONS: The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections.
Asunto(s)
Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Alelos , Secuencia de Aminoácidos , Estudios de Cohortes , Secuencia Conservada/genética , Heterogeneidad Genética , VIH-1/fisiología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Análisis Multivariante , Péptidos/inmunología , Perú , Especificidad de la Especie , Carga Viral/inmunología , Replicación Viral/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.
Asunto(s)
Péptido Hidrolasas/metabolismo , Taenia saginata/enzimología , Taenia solium/enzimología , Animales , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/metabolismo , Quimotripsina/inmunología , Quimotripsina/metabolismo , Activación Enzimática , Humanos , Pancreatina/metabolismo , Péptido Hidrolasas/inmunología , Especificidad por Sustrato , Taenia saginata/inmunología , Taenia saginata/fisiología , Taenia solium/inmunología , Taenia solium/fisiología , Teniasis/parasitologíaRESUMEN
Infections due to Taenia solium in humans (taeniasis/cysticercosis) remain a complex health problem, particularly in developing countries. We identified two oncosphere proteins that might protect the porcine intermediate host against cysticercosis and therefore help prevent disease in humans. One of these proteins was further identified by two-dimensional gel electrophoresis and micro-sequencing. The gene encoding this protective protein was also identified, cloned and characterized. The native 31.5 kDa protein Tso31 has four variants at the cDNA level. The longest sequence from which the others seem to derive, encodes a 253 amino acid peptide. The predicted protein has a molecular weight of 25.1 kDa, one putative N-glycosylation site, two fibronectin type III domains, and one C terminal transmembrane domain. The gene structure of the protein consists of four exons and three introns. The finding of one gene and four different cDNAs for Tso31 suggests the existence of a possible mechanism of differential splicing in this parasite. The Tso31 protein is exclusive to T. solium oncospheres with a putative protein structure of an extra-cellular receptor-like protein. The Tso31 protein was expressed as a recombinant protein fused to GST and tested in a vaccine to determine its effectiveness in protecting pigs against cysticercosis. Only two pigs out of eight vaccinated were protected and although the total median number of cyst decreased in vaccinated pigs compared to controls this decrease was not statistically significant (P = 0.09).
Asunto(s)
Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Taenia solium/inmunología , Secuencias de Aminoácidos/genética , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Antígenos Helmínticos/aislamiento & purificación , Sitios de Unión/genética , Clonación Molecular , Cisticercosis/prevención & control , ADN Complementario/aislamiento & purificación , ADN de Helmintos/química , ADN de Helmintos/genética , Electroforesis en Gel Bidimensional , Glicosilación , Proteínas del Helminto/química , Proteínas del Helminto/genética , Proteínas del Helminto/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Empalme del ARN/fisiología , Análisis de Secuencia de ADN , Análisis de Secuencia de Proteína , Porcinos , Taenia solium/genética , Vacunas de Subunidad/inmunologíaRESUMEN
The specific mechanisms underlying Taenia solium oncosphere adherence and penetration in the host have not been studied previously. We developed an in vitro adhesion model assay to evaluate the mechanisms of T. solium oncosphere adherence to the host cells. The following substrates were used: porcine intestinal mucosal scrapings (PIMS), porcine small intestinal mucosal explants (PSIME), Chinese hamster ovary cells (CHO cells), epithelial cells from ileocecal colorectal adenocarcinoma (HCT-8 cells), and epithelial cells from colorectal adenocarcinoma (Caco-2 cells). CHO cells were used to compare oncosphere adherence to fixed and viable cells, to determine the optimum time of oncosphere incubation, to determine the role of sera and monolayer cell maturation, and to determine the effect of temperature on oncosphere adherence. Light microscopy, scanning microscopy, and transmission microscopy were used to observe morphological characteristics of adhered oncospheres. This study showed in vitro adherence of activated T. solium oncospheres to PIMS, PSIME, monolayer CHO cells, Caco-2 cells, and HCT-8 cells. The reproducibility of T. solium oncosphere adherence was most easily measured with CHO cells. Adherence was enhanced by serum-binding medium with >5% fetal bovine serum, which resulted in a significantly greater number of oncospheres adhering than the number adhering when serum at a concentration less than 2.5% was used (P < 0.05). Oncosphere adherence decreased with incubation of cells at 4 degrees C compared with the adherence at 37 degrees C. Our studies also demonstrated that T. solium oncospheres attach to cells with elongated microvillus processes and that the oncospheres expel external secretory vesicles that have the same oncosphere processes.