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One major unmet need is improving the sensitivity of immune-diagnostic assays. This is particularly important in the field of biomarker discoveries and monitoring. We have established a novel signal amplification probe system enabling a highly sensitive target detection platform to be used in immuno-assays. The probe consists of a double stranded DNA that can carry a large number of signaling elements such as biotin or fluorescent molecules. The DNA probe anchors to the recognition unit, whether an antibody or an aptamer, by covalent conjugation or by a simple and rapid molecular association process. Binding curves obtained by using the DNA amplification probe are dose dependent and linear over a wide range of antigen concentration. The optimal slopes are characterized by high signals and low background increasing the assay sensitivity and reducing the limit of detection by up to 10-fold compared to biotinylated antibodies commonly used in ELISA systems. When using aptamers in combination with the amplification probe for antigen recognition, the limit of detection is comparable to that obtained by biotinylated antibodies. Biotin labeled aptamers practically cannot be used for detection of low target levels. The DNA amplification probe system enables to expand the range of diagnostic assays including clinical samples and meet research needs.
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Anticuerpos/aislamiento & purificación , Aptámeros de Nucleótidos/metabolismo , Sondas de ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Anticuerpos/inmunología , Anticuerpos/metabolismo , Especificidad de Anticuerpos , Aptámeros de Nucleótidos/genética , Biotinilación , Sondas de ADN/genética , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Límite de Detección , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas c-sis/inmunología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Reproducibilidad de los Resultados , Trombina/inmunología , Trombina/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
OBJECTIVE: To evaluate safety and efficacy of DiaPep277 in preserving ß-cell function in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: DIA-AID 1 is a multinational, phase 3, balanced-randomized, double-blind, placebo-controlled, parallel-group clinical study. Newly diagnosed patients (N = 457, aged 16-45 years) were randomized to subcutaneous injections of DiaPep277 or placebo quarterly for 2 years. The primary efficacy end point was the change from baseline in the area under the glucagon-stimulated C-peptide curve. Secondary end points were the change from baseline in mixed-meal stimulated C-peptide secretion and in fasting C-peptide and achieving target HbA1c ≤7% (≤53 mmol/mol). Partial remission (target HbA1c on insulin ≤0.5 units/kg/day) and hypoglycemic event rate were exploratory end points. RESULTS: DiaPep277 was safe and well tolerated. Significant preservation of C-peptide secretion was observed in the DiaPep277-treated group compared with the placebo (relative treatment effects of 23.4%, P = 0.037, and 29.2%, P = 0.011, in the modified intent-to-treat [mITT] and per-protocol [PP] populations, respectively). The mixed-meal stimulation failed to distinguish between the groups. There was a trend toward efficacy in fasting C-peptide levels, though not statistically significant. Significantly more DiaPep277-treated than placebo-treated patients maintained target HbA1c (mITT 56% versus 44%, P = 0.03; PP 60% versus 45%, P = 0.0082) and entered partial remission (mITT 38% versus 29%, P = 0.08; PP 42% versus 30%, P = 0.035). DiaPep277 treatment reduced the relative hypoglycemic event risk (mITT by 20%; PP by 28%). CONCLUSIONS: DiaPep277 safely contributes to preservation of ß-cell function and to improved glycemic control in patients with type 1 diabetes.
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OBJECTIVE: Endogenous insulin secretion, measured by C-peptide area under the curve (AUC), can be tested using both the glucagon stimulation test (GST) and the mixed-meal tolerance test (MMTT). This study compares these two stimulation methods using long-term data from patients newly diagnosed with type 1 diabetes or with latent autoimmune diabetes. RESEARCH DESIGN AND METHODS: A recently completed phase 3 intervention study with DiaPep277 demonstrated improved glycemic control and a significant treatment effect of glucagon-stimulated C-peptide secretion. Unexpectedly, MMTT failed to detect differences between the treated and control groups. Data from 343 patients in two balanced-randomized, double-blind, placebo-controlled, parallel-group trials of DiaPep277 were used to compare and correlate between GST- and MMTT-derived C-peptide AUC. Pearson's correlations were calculated for absolute C-peptide AUC at baseline and 12 and 24 months and for long-term changes in AUC (AUC). RESULTS: The absolute AUC values obtained at any single time point by the two tests were well correlated in both data sets (r = 0.74-0.9). However, the correlations between the AUC were much weaker (r = 0.39-0.58). GST-stimulated C-peptide secretion was stable over the fasting glucose range permitted for the test (4-11.1 mmol/L), but MMTT-stimulated C-peptide secretion decreased over the same range, implying differences in sensitivity to glucose. CONCLUSIONS: Measurement of long-term changes in stimulated C-peptide, reflecting endogenous insulin secretion, during the course of intervention trials may be affected by the method of stimulation, possibly reflecting different sensitivities to the physiological status of the tested subject.
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Chaperonina 60/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Alimentos , Fármacos Gastrointestinales/farmacología , Glucagón/farmacología , Hipoglucemiantes/uso terapéutico , Fragmentos de Péptidos/uso terapéutico , Adolescente , Adulto , Área Bajo la Curva , Glucemia/análisis , Glucemia/metabolismo , Péptido C/metabolismo , Método Doble Ciego , Ayuno/sangre , Femenino , Glucosa/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Masculino , Comidas , Persona de Mediana Edad , Adulto JovenRESUMEN
UNLABELLED: Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. CONCLUSION: These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections.
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Anticuerpos Monoclonales/farmacología , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Virión/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Células Cultivadas , ADN Viral/sangre , Epítopos/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Hepatitis B Crónica , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hepatocitos/virología , Humanos , Modelos TeóricosRESUMEN
BACKGROUND: The aim of this study was to evaluate the effect of an anti-flagellin sub-type monoclonal antibody (anti-fla-a) on Pseudomonas aeruginosa infection in a mouse burn model and to assay bacterial dissemination and invasiveness. METHODS: After immediate post-burn infection with P. aeruginosa, mortality and morbidity (daily weight changes) were monitored in mice treated with anti-fla-a as compared to untreated mice. Bacterial dissemination and invasiveness were monitored by bacterial counts at the burn site and spleen. Three different timing regimens for anti-fla-a treatment were studied: (a) prophylaxis (pre-infection), (b) therapeutic (post-infection), and (c) combined mode. RESULTS: Combined regimen of anti-fla-a markedly improved survival of mice infected with P. aeruginosa from 6% to 96% (p<0.0001), similar to treatment with Imipenem. Furthermore, a significant improvement in survival was obtained when anti-fla-a was given prior to (75% survival) or post-infection (50% survival). It reduced bacterial load in the spleen (p=0.01), preventing bacterial sepsis. CONCLUSION: Anti-fla-a is effective in reducing mortality and morbidity in murine P. aeruginosa-infected burn model.
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Anticuerpos Monoclonales/uso terapéutico , Quemaduras/microbiología , Flagelina/inmunología , Infecciones por Pseudomonas/tratamiento farmacológico , Sepsis/microbiología , Piel/patología , Animales , Especificidad de Anticuerpos , Quemaduras/tratamiento farmacológico , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Sepsis/tratamiento farmacológico , Infección de Heridas/microbiologíaRESUMEN
BACKGROUND/AIMS: HCV-AB68, a human monoclonal antibody against the envelope protein of hepatitis C virus (HCV), neutralizes HCV in cell-culture and in the HCV-Trimera mouse model. A Phase 1 clinical trial was designed to test safety, tolerability, and antiviral activity of HCV-AB68 in patients with chronic HCV-infection. METHODS/RESULTS: Single doses of HCV-AB68, 0.25-40 mg, administered to 15 patients were well tolerated with no moderate or serious adverse events (SAEs) reported. In six patients, HCV-RNA levels transiently decreased by 2- to 100-fold immediately following infusion and rebound to baseline in 24-48 h. Multiple doses of HCV-AB68, 10-120 mg, were administered to 25 patients. Doses were given weekly for 3 weeks, then 3x a week during the fourth week, after which patients were followed for 3 months. No drug-related SAEs were reported and no specific pattern of adverse events was evident. Eight out of 25 patients had at least a 1-log reduction and 17 had at least a 0.75-log reduction in HCV-RNA levels from baseline at one or more time points following HCV-AB68 infusion. CONCLUSIONS: These data support the investigation of HCV-AB68 in the prevention of recurrent HCV-infection in patients who had received hepatic allografts for end-stage liver disease.
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Anticuerpos Monoclonales/uso terapéutico , Antivirales/uso terapéutico , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/uso terapéutico , Hepatitis C Crónica/terapia , Adolescente , Adulto , Anciano , Animales , Anticuerpos Monoclonales/efectos adversos , Antivirales/efectos adversos , Secuencia de Bases , Cartilla de ADN/genética , Tolerancia a Medicamentos , Femenino , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/efectos adversos , Hepatitis C Crónica/virología , Humanos , Masculino , Ratones , Persona de Mediana Edad , ARN Viral/sangre , ARN Viral/genética , SeguridadRESUMEN
A randomized, double-blind, dose-escalation study evaluated the safety and efficacy of hepatitis C virus (HCV)-Ab(XTL)68, a neutralizing, high-affinity, fully human, anti-E2 monoclonal antibody, in 24 HCV-positive patients undergoing liver transplantation. HCV-Ab(XTL)68 or placebo was administered at doses from 20-240 mg as 2-4 infusions during the first 24 hours after transplantation, followed by daily infusions for 6 days, weekly infusions for 3 weeks, and either 2 or 4 weekly infusions for 8 weeks. Serum concentrations of total anti-E2 obtained during daily infusions of 120-240 mg HCV-Ab(XTL)68 were 50-200 microg/mL above concentrations in the placebo group. Median serum concentration of HCV RNA dropped below baseline in all groups immediately after transplantation. On day 2, median change from baseline in HCV RNA was -1.8 and -2.4 log in the 120-mg and 240-mg groups, respectively, compared with -1.5 log with placebo. The difference was lost after day 7 when the dosing frequency was reduced. The coincidence of increases in anti-E2 with decreases in HCV RNA concentration indicate that the dose-related changes in HCV RNA concentration were a result of HCV-Ab(XTL)68 administration in the 120- and 240-mg groups. The overall incidence of nonfatal serious adverse events was higher with placebo (60%) vs. all active treatments combined (42%). In conclusion, HCV-Ab(XTL)68 may decrease serum concentrations of HCV RNA in patients after liver transplantation. Studies evaluating more frequent daily dosing at doses >120 mg are necessary to investigate sustained viral suppression in this population.
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Anticuerpos Monoclonales/farmacología , Hepacivirus/inmunología , Hepatitis C/prevención & control , Inmunoglobulinas/farmacología , Trasplante de Hígado/inmunología , Adulto , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Hepatitis C/inmunología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
We have evaluated the effect of antibodies against the Candida albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a potential immunotherapeutic treatment for acute invasive candidiasis in a murine model of infection. Three different approaches were assayed: (i) active immunization of mice using recombinant His-tagged GAPDH, (ii) treatment of fungal yeast cells with anti-GAPDH antibodies prior to infection, and (iii) passive transfer of polyclonal anti-GAPDH antibodies. Results showed that all three approaches, although tending to show a slight beneficial effect in some instances, fail to have a relevant and statistically significant effect on the infection course, determined by survival curves and fungal burden in kidneys. This suggests that the cell wall-associated GAPDH of C. albicans, despite its potential role in virulence, does not appear to be a suitable target protein for the development of immunotherapeutic strategies against candidiasis, although further studies may be required to confirm this observation.
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Anticuerpos Antifúngicos/uso terapéutico , Candida albicans/inmunología , Candidiasis/terapia , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Animales , Anticuerpos Antifúngicos/inmunología , Antígenos Fúngicos/inmunología , Candida albicans/enzimología , Candidiasis/microbiología , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Inmunización , Inmunización Pasiva , Ratones , Ratones Endogámicos ICR , ConejosRESUMEN
BACKGROUND: In an era of increasing drug resistance, immunotherapy is a desirable treatment against Pseudomonas aeruginosa infections. The flagellum, which is an important pseudomonal virulence factor, was targeted for immunotherapy. The aim of the study was to evaluate the efficacy of polyclonal immunotherapy targeted against the N-terminal of flagellin (anti-N'-fla-b) for treating severe P. aeruginosa infection in a murine burn wound model. METHODS: Groups of 12 mice were infected (subeschar) with P. aeruginosa strain PA01, and were treated either with systemic anti-N'-fla-b immunoglobulin G (IgG), nonspecific IgG, or imipenem. The control groups included mice with burn alone, mice with untreated infected burn, and mice without burn infected with P. aeruginosa. Three separate regimens were examined: prophylaxis (preinfection), therapeutic (postinfection), and combined. The efficacy of anti-N'-fla-b was evaluated by monitoring the mortality and morbidity (relative weight loss) during a period of 2 weeks. RESULTS: Anti-N'-fla-b IgG immunotherapy significantly decreased the mortality rate of infected burned mice followed by severe P. aeruginosa infection. The mortality rate in the anti-N'-fla-b-treated groups ranged from 0 to 17 percent compared with 58 to 83 percent in nontreated groups infected with 2 to 5 x 10(6) colony-forming units of P. aeruginosa (p < 0.05). The mortality rate in the anti-N'-fla-b-treated groups was similar to that of groups treated with imipenem. The three tested regimens yielded similar results. Morbidity paralleled survival results. Histopathologic examination revealed an earlier reepithelialization of the infected wound in the anti-N'-fla-b-treated mice compared with untreated mice. CONCLUSION: Immunotherapy with anti-N'-fla-b IgG, given either as prophylaxis or therapeutically, effectively reduced mortality and morbidity and improved wound healing in a severely P. aeruginosa-infected murine burn model.
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Quemaduras/complicaciones , Flagelina/inmunología , Inmunoglobulina G/uso terapéutico , Infecciones por Pseudomonas/terapia , Pseudomonas aeruginosa/inmunología , Infección de Heridas/terapia , Animales , Antibacterianos/uso terapéutico , Quemaduras/mortalidad , Modelos Animales de Enfermedad , Femenino , Flagelina/antagonistas & inhibidores , Imipenem/uso terapéutico , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Infecciones por Pseudomonas/mortalidad , Infecciones por Pseudomonas/prevención & control , Conejos , Cicatrización de Heridas , Infección de Heridas/mortalidadRESUMEN
A simple reproducible and versatile small animal model for hepatitis B virus (HBV) infection is still unavailable. We have generated a simple transient liver-targeted transgenic mouse. Hydrodynamics tail vein injection of a head-to-tail dimer of adw HBV genome (pHBVadwHTD) into immunocompetent mice generated HBsAg and HBeAg expression in both serum and hepatocytes, followed by seroconversion. The injection of pHBVadwHTD into SCID mice generated prolonged HBsAg and HBeAg antigenemia and HBV viremia. Our results demonstrate that hydrodynamic injection of naked DNA could support the generation of HBV particles. We used this model for the assessment of anti-viral agents. Administration of our human monoclonal antibodies, HBV-Ab17(XTL) and HBV-Ab19(XTL), as well as Lamivudine (3TC) treatment suppressed HBV viremia. The model presented herein supports long and stable expression of HBV and will enable determination of various biological questions related to HBV life cycle, mutants and could enhance the development of anti-viral reagents.
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Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.
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Anticuerpos Monoclonales/uso terapéutico , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/uso terapéutico , Hepatitis C/prevención & control , Trasplante de Hígado/efectos adversos , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Animales , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Datos de Secuencia Molecular , Pruebas de Neutralización , Recurrencia , Análisis de Secuencia de ADNRESUMEN
The goal of this study was to investigate if antibodies raised against N'-terminal Pseudomonas aeruginosa (Pa) flagellin could afford protection in two lethal mouse models of Pa infection. To that end, rabbit polyclonal antibodies were generated against the N'-terminal domains (amino acids 1-156) of recombinant Pa01 or Salmonella muenchen flagellins, termed anti-N'-fla-b and anti-N'-fla-Sm, respectively. In vitro, anti-N'-fla-b but not anti-N'-fla-Sm IgG specifically recognized recombinant and Pa endogenous flagellin type b proteins, total bacterial lysates of Pa type b, and inhibited Pa01 invasion into A549 cells. In vivo, administration of anti-N'-fla-b afforded a remarkable improvement in survival in lethal peritonitis (90% vs. 12% in control; p<0.001) and burn infection (83% vs. 8-17% in control groups; p<0.005) Pa models. These findings would suggest that the N'-terminal domain of Pa flagellin harbors critically important bioactive domains and that an antibody-targeted, neutralization approach directed at this region could provide a novel therapeutic strategy to combat Pa infection.
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Anticuerpos Antibacterianos/inmunología , Modelos Animales de Enfermedad , Flagelina/química , Flagelina/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Femenino , Ratones , Datos de Secuencia Molecular , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/química , Conejos , Alineación de Secuencia , Tasa de SupervivenciaRESUMEN
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.
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Clonación Molecular/métodos , ADN Complementario , Genes de Inmunoglobulinas , Oligonucleótidos/química , Biblioteca de Péptidos , Adolescente , Adulto , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Biotecnología/métodos , Candida albicans/enzimología , Candida albicans/inmunología , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Cryptic hepatitis C virus (HCV) infection relates to patients infected chronically with HCV that are seronegative but have HCV-RNA. These patients are not identified by the standard serological tests for HCV, which are based on detection of antibodies to core, NS3 and NS5 antigens. They will, therefore, be wrongly diagnosed as non-infected, and are considered as a potential risk for others. Cryptic HCV infection in dialysis units occurs frequently and, due to medical procedures, is a major factor for contracting the virus when unrecognised. This study was conducted in order to assess the humoral immune responses to E2-antigen in sera of patients infected chronically with HCV. Recombinant E2 protein in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) were used to test the occurrence of anti-E2 antibodies in the sera of patients from the liver clinic and of dialysis patients. The presence of E2 antibodies was found to be correlated with the presence of HCV-RNA and with viral load. Antibodies to the E2 protein could be detected in as many as 30% of the sera from dialysis patients with cryptic HCV infection (HCV-RNA only). The results suggest that detection of anti-E2 antibodies may enhance significantly HCV serological standard testing; especially among patients on dialysis, and that antibodies to envelope E2 protein appear to depend on and correlate with the presence of HCV particles.
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Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C Crónica/virología , Diálisis Renal , Proteínas del Envoltorio Viral/inmunología , Viremia/virología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Antígenos de la Hepatitis C/genética , Antígenos de la Hepatitis C/inmunología , Hepatitis C Crónica/inmunología , Humanos , ARN Viral/sangre , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/genética , Carga Viral , Viremia/inmunologíaRESUMEN
Antibodies have the potential to be immunotherapeutic agents, used either as stand-alone therapy or as an adjunct for managing chronic viral infection. In addition, antibodies may be used prophylactically in individuals who have been accidentally exposed to hepatitis B virus (HBV) or hepatitis C virus (HCV), or to prevent re-infection of the liver in patients who have undergone liver transplantation. Human monoclonal antibodies to HBV and HCV were generated and their ability to reduce viral load was tested in different animal model systems, the Trimera mouse model and HBV-carrier chimpanzees. These antibodies were further developed and are currently being studied in clinical trials for chronic HBV or HCV and in liver transplant patients. The antibodies were shown to be safe, tolerable and could significantly reduce viral load. Their ability to inhibit HCV re-infection in the transplanted liver is being evaluated.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antihepatitis/inmunología , Anticuerpos Antihepatitis/uso terapéutico , Hepatitis B/terapia , Hepatitis C/terapia , Inmunoterapia , Animales , Hepatitis B/inmunología , Hepatitis C/inmunología , Humanos , Ratones , Pan troglodytesRESUMEN
A Trimera mouse is constructed through a three-step process. Firstly, a normal mouse host is rendered immuno-incompetent by a lethal split-dose total body irradiation. Secondly, the myeloid and erythroid lineages are reconstituted by transplantation of bone marrow cells from a genetically immune-deficient mouse donor. Thirdly. the resulting preconditioned mouse is transplanted with human cells or tissues that can be maintained in the foreign, yet supporting, environment for a considerable period of time. Immunization of Trimera mice, engrafted with human immune cells, induces a strong human immune response, thereby enabling generation of human therapeutic monoclonal antibodies (mAbs) via hybridoma technology. Transplantation of infected human tissue into the preconditioned mice results in the creation of Trimera mouse models for human diseases.
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Anticuerpos Monoclonales/biosíntesis , Modelos Animales de Enfermedad , Ratones Mutantes , Animales , Hepatitis B/inmunología , Hepatitis B/patología , Virus de la Hepatitis B/inmunología , Hepatitis C/inmunología , Hepatitis C/patología , Ratones , Neoplasias/inmunología , Neoplasias/patología , RatasRESUMEN
Treatment of chronic hepatitis B virus (HBV) infection with interferon alfa and lamivudine is characterized by lack of viral clearance, loss of response, or emergence of drug-resistant mutants. Thus, new and multiple drug approaches are needed. We have developed two fully human monoclonal antibodies, directed against different epitopes of hepatitis B surface antigen (HBsAg) that bind to all major HBV subtypes. A phase I clinical study was conducted to evaluate the safety, tolerability, and efficacy of a mixture of these two monoclonal antibodies, HBV-AB(XTL). A total of 27 chronic HBV patients were enrolled. In part A of the study 15 patients in 5 cohorts received a single intravenous infusion of antibodies with doses ranging from 0.26 mg (260 IU) to 40 mg (40,000 IU). All patients completed 16 weeks of follow-up. In the second part of the study (part B), 12 patients in 4 cohorts received 4 weekly infusions of 10, 20, 40, or 80 mg each of HBV-AB(XTL) and were followed for 4 additional weeks. Administration of antibodies was well tolerated. Patients administered doses at an Ab:Ag molar ratio of 1:2 to 1:20 showed a rapid and significant decrease in HBsAg to undetectable levels, with a corresponding reduction of HBV-DNA levels. In part B, HBV-AB(XTL) induced a significant reduction in both HBsAg and HBV-DNA levels repeatedly after administration. In conclusion, these data suggest that HBV-AB(XTL) binds HBV particles and reduces serum viral titers and HBsAg levels. HBV-AB(XTL) could be combined with other monotherapies that are currently used to treat HBV carriers.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos contra la Hepatitis B/administración & dosificación , Antígenos de Superficie de la Hepatitis B/inmunología , Hepatitis B Crónica/terapia , Adulto , Anciano , Anticuerpos Monoclonales/efectos adversos , ADN Viral/análisis , Femenino , Antígenos de Superficie de la Hepatitis B/análisis , Hepatitis B Crónica/virología , Humanos , Inmunoglobulinas/uso terapéutico , Masculino , Persona de Mediana EdadRESUMEN
The lack of small-animal models that are suitable for evaluation of agents used to treat infection with hepatitis C virus (HCV) severely hinders the assessment of potential new therapies for the disease. This study created such a model, termed the "HCV-Trimera" model. The HCV-Trimera model was developed by using lethally irradiated mice, reconstituted with SCID mouse bone marrow cells, in which human liver fragments infected ex vivo with HCV had been transplanted. Viremia (positive-strand HCV RNA levels) in HCV-Trimera mice peaked at approximately day 18 after liver transplantation, and an infection rate of 85% was reached. Viral replication in liver grafts was evidenced by the presence of specific negative-strand HCV RNA. The usefulness of this model for evaluation of anti-HCV agents was demonstrated by the ability of a small molecule (an HCV internal ribosomal entry site inhibitor) and an anti-HCV human monoclonal antibody (HCV AB(XTL)68) to reduce virus loads in HCV-Trimera mice in a dose-dependent manner.