RESUMEN
Immunogenicity assessment of fully human monoclonal antibody-based biotherapeutics requires sensitive and specific ligand binding assays. One of the components of specificity is the depletion of signal by a relevant biotherapeutic that is commonly based on an arbitrary depletion criterion of inhibition of the original response or reduction of the signal below the screening assay cut point (ACP). Hence, there is a need to develop a statistically derived physiologically relevant specificity criterion. We illustrate an optimization approach to determine the concentration of biotherapeutic required for the specificity evaluation. Naïve donor sample sets with and without circulating drug and antitherapeutic/drug antibody (ADA) were prepared. Next, a depletion cut point (DCP) using naïve and ADA-containing donor sets with the optimized biotherapeutic concentration was evaluated. A statistically derived design of experiment was used to establish a validated DCP. A reliable DCP requires naïve (no ADA) donors treated only with an optimized concentration of biotherapeutic. The additional DCPs generated using two distinct concentrations of ADA-spiked sample sets led to a physiologically irrelevant criterion that was not necessarily representative of real-time samples. This increased the risk of false positives or negatives. In this study, well-defined bioanalytical and statistical methods were employed to validate a DCP to confirm the presence of biotherapeutic specific ADA in human serum samples. A physiologically relevant and effective strategy to confirm specificity in immune reactive samples, especially those that are close to the ACP, is proposed through this study.
Asunto(s)
Anticuerpos Monoclonales/sangre , Fenómenos Inmunogenéticos/fisiología , Inmunoglobulina G/sangre , Análisis por Matrices de Proteínas/normas , Eliminación de Secuencia/inmunología , Terapia Biológica/normas , Femenino , Humanos , Fenómenos Inmunogenéticos/efectos de los fármacos , Masculino , Análisis por Matrices de Proteínas/estadística & datos numéricos , Eliminación de Secuencia/genéticaRESUMEN
Viral vectors have provided effective methods for in vivo gene delivery for therapeutic purposes. The ability of viruses to infect a wide variety of cell types in vivo has been exploited for several applications, such as liver, lung, muscle, brain, eye and many others. Immune responses directed towards the viral capsids and the transgene products have severely affected the ability of these vectors to induce long-term gene expression. This paper reviews the influence of viral vectors on antigen-presenting cells (APC), which are central to the induction of innate as well as adaptive immune responses. In this respect, we have focused on adenovirus and adeno-associated viruses because of the polar responses these vector systems induce in vivo. While adenovirus vector can induce significant inflammatory responses, adeno-associated viral vectors are characterized by their inability to consistantly induce immune responses to the transgene product. Understanding the mechanism of infection, transduction and activation of APC by viral vectors will provide strategies to develop safe vectors and prevent immune responses in gene therapies.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Adenoviridae/inmunología , Células Presentadoras de Antígenos/inmunología , Dependovirus/inmunología , Terapia Genética/efectos adversos , Vectores Genéticos/inmunología , Animales , Células Presentadoras de Antígenos/virología , Linfocitos B/inmunología , Ingeniería Genética , Vectores Genéticos/administración & dosificación , Humanos , Activación de Linfocitos , Transducción Genética , Transgenes , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.
Asunto(s)
Dependovirus/genética , Terapia Genética , Vectores Genéticos , Músculos/metabolismo , Animales , Anticuerpos Antivirales/sangre , Dependovirus/clasificación , Dependovirus/inmunología , Humanos , Hibridación Genética , Inmunización , Ratones , Ratones Endogámicos C57BL , SerotipificaciónRESUMEN
We investigated the cellular basis for secretion of inflammatory cytokines in mice following intravenous administration of adenoviral vectors (Ad). Serum inflammatory cytokines including interleukin-6 (IL-6), IL-12, and tumor necrosis factor-alpha (TNF-alpha) were detected as early as 6 h following intravenous injection of Ad-expressing Escherichia coli beta-galactosidase (Ad-lacZ). Ad-lacZ readily accumulated in the splenic marginal zone 1 h after intravenous infusion, where both dendritic cells (DCs) and macrophages were transduced and activated within 6 h. Flow cytometric analyses showed that the expression of Ia and CD86 antigens was markedly enhanced on splenic DCs indicating their activation in vivo by Ad-lacZ. Upon ex vivo culture, these early-activated splenic DCs spontaneously produced high levels of IL-6 and IL-12. By contrast, activated splenic macrophages spontaneously secreted only IL-6. Elimination of tissue macrophages and splenic DCs in vivo considerably reduced the early release of IL-12, IL-6, and TNF-alpha and significantly blocked the specific cellular immune response to Ad and the transgene product in vivo. Our findings indicate that preferential activation of DCs and macrophages may account for Ad-triggered acute inflammatory response in vivo in mice. Moreover, DCs and macrophages may play different roles in this process in terms of their abilities to produce distinct patterns of inflammatory cytokines.
Asunto(s)
Adenoviridae/genética , Citocinas/biosíntesis , Células Dendríticas/metabolismo , Inflamación , Macrófagos/metabolismo , Animales , Antígenos CD/biosíntesis , Antígeno B7-2 , Células Cultivadas , Relación Dosis-Respuesta a Droga , Escherichia coli/enzimología , Citometría de Flujo , Inmunohistoquímica , Interleucina-12/biosíntesis , Interleucina-6/biosíntesis , Operón Lac , Liposomas/metabolismo , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Bazo/citología , Bazo/metabolismo , Linfocitos T Citotóxicos/metabolismo , Factores de Tiempo , Distribución Tisular , Transgenes , Factor de Necrosis Tumoral alfa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
Adenovirus vectors have been studied as vehicles for gene transfer to skeletal muscle, an attractive target for gene therapies for inherited and acquired diseases. In this setting, immune responses to viral proteins and/or transgene products cause inflammation and lead to loss of transgene expression. A few studies in murine models have suggested that the destructive cell-mediated immune response to virally encoded proteins of E1-deleted adenovirus may not contribute to the elimination of transgene-expressing cells. However, the impact of immune responses following intramuscular administration of adenovirus vectors on transgene stability has not been elucidated in larger animal models such as nonhuman primates. Here we demonstrate that intramuscular administration of E1-deleted adenovirus vector expressing rhesus monkey erythropoietin or growth hormone to rhesus monkeys results in generation of a Th1-dependent cytotoxic T-cell response to adenovirus proteins. Transgene expression dropped significantly over time but was still detectable in some animals after 6 months. Systemic levels of adenovirus-specific neutralizing antibodies were generated, which blocked vector readministration. These studies indicate that the cellular and humoral immune response generated to adenovirus proteins, in the context of transgenes encoding self-proteins, hinders long-term transgene expression and readministration with first-generation vectors.
Asunto(s)
Adenoviridae/genética , Proteínas E1 de Adenovirus/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Adenoviridae/inmunología , Animales , Anticuerpos Antivirales/sangre , Citocinas/análisis , Eritropoyetina/sangre , Eritropoyetina/genética , Eliminación de Gen , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Hormona del Crecimiento/sangre , Hormona del Crecimiento/genética , Inyecciones Intramusculares , Activación de Linfocitos , Macaca mulatta , Pruebas de Neutralización , Linfocitos T Citotóxicos/inmunología , TransfecciónRESUMEN
Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.
Asunto(s)
Adenovirus Humanos/inmunología , Expresión Génica , Vectores Genéticos/inmunología , Polietilenglicoles , Succinimidas/inmunología , Sulfonas/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Línea Celular , Genes Reporteros , Humanos , Inmunidad Celular , Inmunocompetencia , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Pulmón/inmunología , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/virología , beta-Galactosidasa/genéticaRESUMEN
Recombinant adeno-associated virus (rAAV) vectors allow efficient gene transfer and expression in the muscle; therefore, rAAVs represent a potential gene therapy vector for muscular dystrophies. For further investigations, we used a mouse muscular dystrophy model (gsg(-/-) mice) gamma-sarcoglycan, a subunit of the dystrophin-glycoprotein complex, is missing. gsg(-/-) mice develop progressive dystrophy representative of a severe human phenotype disease. We previously showed high levels and stable expression of gamma-sarcoglycan in myofibers after direct muscle injection into gsg(-/-) mice of a recombinant AAV vector (AAV.dMCK.gSG) carrying the gamma-sarcoglycan cDNA driven by a muscle-specific promoter (truncated version of muscle creatine kinase). Here, we show that when gamma-sarcoglycan expression is driven by the ubiquitous cytomegalovirus (CMV) promoter (AAV.CMV.gSG), lower levels of transgene expression are observed and are associated with a humoral response to gamma-sarcoglycan. When using an rAAV vector, expressing the highly immunogenic product gamma-galactosidase under the CMV promoter (AAV.CMV.LacZ), we measured a strong cellular and humoral immune response to the transgene after intramuscular injection into gsg(-/-) mice. This study suggests that restriction of transgene expression to the muscle is an important criterion for the treatment of muscular dystrophies and will aid in the design of protocols for gene therapy.
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Dependovirus/genética , Técnicas de Transferencia de Gen , Glicoproteínas de Membrana/biosíntesis , Músculo Esquelético/metabolismo , Distrofias Musculares/terapia , Regiones Promotoras Genéticas , Animales , Células Presentadoras de Antígenos/inmunología , Western Blotting , Creatina Quinasa/genética , Células Dendríticas/inmunología , Distrofina/biosíntesis , Vectores Genéticos , Humanos , Técnicas para Inmunoenzimas , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Distrofias Musculares/genética , Distrofias Musculares/inmunología , Distrofias Musculares/metabolismo , Sarcoglicanos , Linfocitos T Citotóxicos , Transducción Genética , beta-Galactosidasa/metabolismoRESUMEN
Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Dependovirus/genética , Dependovirus/aislamiento & purificación , Terapia Genética/métodos , Vectores Genéticos/aislamiento & purificación , Animales , Reactores Biológicos , Western Blotting , Línea Celular , Centrifugación por Gradiente de Densidad , Citocinas/metabolismo , Dependovirus/ultraestructura , Electroforesis en Gel de Poliacrilamida , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Músculos/virología , Tinción con Nitrato de Plata , Tibia/virología , Factores de Tiempo , Transducción Genética , Transfección , Rayos UltravioletaRESUMEN
Recombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding beta-galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ 10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L(-/-)) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a beta-galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZ transduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.
Asunto(s)
Antígenos CD40/inmunología , Células Dendríticas/inmunología , Dependovirus/genética , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Células de la Médula Ósea/citología , Células Dendríticas/trasplante , Dependovirus/inmunología , Técnica del Anticuerpo Fluorescente , Técnicas de Transferencia de Gen , Vectores Genéticos , Operón Lac , Ligandos , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/inmunología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Vectors based on adeno-associated viruses (AAV) type 2 show promise for treating chronic diseases because transgene expression appears to be stable. This study evaluated the impact of humoral immunity to the capsid proteins on vector uptake by hepatocytes following an intravascular approach. Route of vector administration in mice had a qualitative effect on antivector B cell responses. Administration of vector into the tail vein resulted in T-cell-dependent (TD) B cell responses that were completely inhibited with depleting CD4 antibody. Delivery of vector into the portal circulation via the spleen yielded B cell response that were partially T cell independent (TI) rendering strategies based on T cell inhibition ineffective in allowing vector readministration. The TI B cell response was short lived in comparison to the TD response. Rhesus monkeys produced a B cell memory response to intraportal vector which appeared to be T cell dependent based on Ig isotypes. Gene therapy strategies that require AAV vector readministration should consider vector biodistribution and its impact on B cell activation.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Dependovirus/genética , Dependovirus/inmunología , Vectores Genéticos , Animales , Linfocitos B/inmunología , Terapia Genética , Humanos , Memoria Inmunológica , Inyecciones , Inyecciones Intravenosas , Hígado , Activación de Linfocitos , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Vena Porta , Bazo , Linfocitos T/inmunologíaRESUMEN
We have previously reported a factor VIII knockout (FVIII KO) mouse model for hemophilia A. Here we demonstrate the presence of nonfunctional heavy chain factor VIII protein in the mouse, making it an excellent model for cross-reacting material (CRM)-positive hemophilia A patients, who express normal levels of a dysfunctional FVIII protein. We attempted to correct these mice phenotypically by transduction of wild-type mouse factor VIII cDNA delivered in an E1/E3-deleted adenoviral vector by tail vein injection. All treated mice displayed initial high-level FVIII expression that diminished after 1 month. Ten of 12 mice administered between 6 x 10(9) and 1 x 10(11) particles/mouse along with anti-CD4 antibody showed long-term FVIII activity (0.03-0.05 IU/ml, equivalent to 3-5% of normal FVIII) that corrected the phenotype. Wild-type murine FVIII was a neo-antigen to the KO mice, generating both cytotoxic and humoral immune responses. Immune suppression with anti-CD4 antibody abrogated these immune responses. These data demonstrate that despite the presence of endogenous FVIII protein the immune system still recognizes a species-specific transgene protein as a neo-antigen, eliciting a cytotoxic T cell response. This phenomenon may exist in the treatment of other genetic disorders by gene therapy.
Asunto(s)
Factor VIII/genética , Terapia Genética , Hemofilia A/terapia , Adenoviridae/genética , Animales , Southern Blotting , Western Blotting , Citotoxicidad Inmunológica , Factor VIII/inmunología , Factor VIII/metabolismo , Terapia Genética/efectos adversos , Vectores Genéticos , Hemofilia A/inmunología , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Linfocitos T/inmunologíaRESUMEN
The interaction between CD40 on B cells and CD40 ligand (CD40L) on activated T cells is important for B-cell differentiation in T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus monkeys in the presence or absence of a transient treatment with a humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated with hu5C8 at the time vector was instilled into the lung. Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM) levels comparable to control animals, but did not secrete IgA or develop neutralizing antibodies; consequently, the animals could be readministered with adenovirus vector expressing alkaline phosphatase. A second study was designed to examine the long-term effects on immune functions of a short course of hu5C8. Acute hu5C8 treatment resulted in significant and prolonged inhibition of the adenovirus-specific humoral response well beyond the time hu5C8 effects were no longer significant. These studies demonstrate the potential of hu5C8 as an immunomodulatory regimen to enable administration of adenoviral vectors, and they advocate testing this model in humans.
Asunto(s)
Adenoviridae/genética , Antígenos CD40/metabolismo , Vectores Genéticos , Pulmón/virología , Glicoproteínas de Membrana/metabolismo , Animales , Anticuerpos Monoclonales/uso terapéutico , Formación de Anticuerpos , Ligando de CD40 , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Terapia Genética , Inmunidad Celular , Macaca mulattaRESUMEN
Subretinal delivery of recombinant adeno-associated virus (rAAV) results in a systemic humoral response in the adult immunocompetent mouse. We characterized this response and determined whether it is possible to readminister rAAV to the subretinal space despite the presence of antibodies to the vector. A systemic humoral response to rAAV capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of rAAV carrying the cDNA encoding green fluorescent protein (GFP), rAAV-GFP. Experiments were performed in cohorts of adult C57BL/6 mice. Assessment of systemic humoral response to viral capsid protein was performed through enzyme-linked immunoabsorbent assay (ELISA) and infection inhibition studies of serum samples 3 weeks after virus delivery. The rAAV-GFP virus was readministered by subretinal injection. GFP expression after subretinal administration was evaluated ophthalmoscopically throughout the course of the experiment and histologically at the termination of the experiment. We observed significant systemic humoral responses to viral capsid protein after subretinal delivery of rAAV. Intradermal injection resulted in a larger humoral response (with a higher percentage of neutralizing antibodies) than subretinal injection. Additional transduction events were observed after readministration of rAAV despite the presence of strong humoral response to the vector.
Asunto(s)
Cápside/inmunología , Dependovirus/genética , Técnicas de Transferencia de Gen , Retina/inmunología , Transducción Genética , Animales , Dependovirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Proteínas Fluorescentes Verdes , Inyecciones , Inyecciones Intradérmicas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Microscopía FluorescenteRESUMEN
Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.
Asunto(s)
Cápside/administración & dosificación , Dependovirus/inmunología , Infecciones por Parvoviridae/inmunología , Animales , Anticuerpos/farmacología , Anticuerpos Antivirales/sangre , Antígenos CD4/genética , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Cápside/biosíntesis , Cápside/genética , Dependovirus/genética , Terapia Genética , Vectores Genéticos , Isotipos de Inmunoglobulinas/sangre , Inyecciones Intramusculares , Macaca mulatta , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Desnudos , Ratones Transgénicos , Infecciones por Parvoviridae/sangre , Infecciones por Parvoviridae/prevención & control , Proteínas Recombinantes/administración & dosificación , Factores de TiempoRESUMEN
CD40 is thought to play a central role in T cell-dependent humoral responses through two distinct mechanisms. CD4+ T helper cells are activated via CD40-dependent Ag presentation in which CD80/CD86 provides costimulation through CD28. In addition, engagement of CD40 on B cells provides a direct pathway for activation of humoral responses. We used a model of adenovirus-mediated gene transfer of beta-galactosidase (lacZ) into murine lung to evaluate the specific CD40-dependent pathways required for humoral immunity at mucosal surfaces of the lung. Animals deficient in CD40L failed to develop T and B cell responses to vector. Activation of Th2 cells, which normally requires CD40-dependent stimulation of APCs, was selectively reconstituted in CD40 ligand-deficient mice by systemic administration of an Ab that is agonistic to CD28. Surprisingly, this resulted in the development of a functional humoral response to vector as evidenced by formation of germinal centers and production of antiadenovirus IgG1 and IgA that neutralized and prevented effective readministration of vector. The CD28-dependent B cell response required CD4+ T cells and was mediated via IL-4. These studies indicate that CD40 signals to the B cells are not necessary for CD4+ Th2 cell-dependent humoral responses to be generated.
Asunto(s)
Linfocitos B/inmunología , Antígenos CD40/genética , Antígenos CD40/metabolismo , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Células Th2/inmunología , Adenoviridae/genética , Adenoviridae/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Linfocitos B/metabolismo , Linfocitos B/virología , Antígenos CD28/fisiología , Antígenos CD40/fisiología , Ligando de CD40 , Separación Celular , Citotoxicidad Inmunológica/genética , Vectores Genéticos/inmunología , Humanos , Interleucina-4/fisiología , Ligandos , Activación de Linfocitos/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Citotóxicos/inmunologíaRESUMEN
A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.
Asunto(s)
Adenoviridae/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/terapia , Terapia Genética , Pulmón/metabolismo , Secuencia de Bases , Fibrosis Quística/inmunología , Sondas de ADN , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Humanos , Pruebas de NeutralizaciónRESUMEN
The early successes of adenoviral vector-mediated gene therapies in the lung have been hampered by the immune response directed against viral proteins and transgene product. Intratracheal administration of adenovirus vector in immune-competent mice transduces bronchioepithelial cells of lung extremely efficiently; however, transgene expression is eliminated within 2 weeks. Extinction of transgene expression has been attributed to infiltrating cytotoxic T lymphocytes (CTLs). Fas-Fas ligand (Fas-FasL) interactions play a critical role in the effector function of the CTL killing. We have previously demonstrated that this interacting pair of molecules plays a critical role in elimination of transgene expression in mouse liver. In this study we investigated the role of Fas-FasL interactions in CTL effector functions in vivo in mouse lung. Analyses of these molecules in lung showed constitutive expression of Fas in bronchiooepithelial cells. On the other hand, FasL was inducible in the bronchioepithelial cells after adenovirus vector treatment. The in vivo role of the Fas-FasL interactions was determined by adoptive transfer of splenocytes from normal immune-competent mice to Fas-deficient mice (B6-lpr); likewise, the function of FasL on CTLs was analyzed by the ability of splenocytes from FasL-deficient mice (B6-gld) to eliminate transgene expression in Rag1-deficient mice. These studies demonstrate that despite expression of Fas and FasL in bronchioepithelial cells and CTLs, these interacting molecules do not play a critical role in elimination of transgene expression in the lung.
Asunto(s)
Adenoviridae/genética , Bronquios/inmunología , Terapia Genética , Vectores Genéticos , Pulmón/metabolismo , Glicoproteínas de Membrana/metabolismo , Linfocitos T Citotóxicos/inmunología , Receptor fas/metabolismo , Animales , Bronquios/citología , Células Epiteliales/inmunología , Proteína Ligando Fas , Expresión Génica , Pulmón/inmunología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Transgenes , Receptor fas/inmunologíaRESUMEN
Vectors based on human adenovirus (Ad) and adeno-associated virus (AAV) are being evaluated for human gene therapy. The response of the host to the vector, in terms of antigen-specific immunity, will play a substantial role in clinical outcome. We have surveyed cohorts of normal subjects and cystic fibrosis patients for pre-existing immunity to these viruses, caused by naturally acquired infections. A number of humoral and cellular assays to adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 2 (AAV2) were performed from serum and peripheral blood mononuclear cells. Virtually all subjects had Ig to Ad5 although only 55% of these antibodies neutralized virus (NAB). Approximately two of three patients demonstrated CD4+ T cells that proliferated to Ad antigens of which most were of the TH1 subset, based on cytokine secretion. A substantially different pattern of immune responses was observed to AAV2. Although virtually all patients had Ig to AAV2, most of these antibodies were not neutralizing (32% NAB) and only 5% of patients had peripheral blood lymphocytes that proliferated in response to AAV2 antigens. These studies demonstrate marked heterogeneity in pre-existing immunity to Ad5 and AAV2 in human populations. The impact of these findings on outcome following gene therapy will require further study.
Asunto(s)
Adenoviridae/inmunología , Anticuerpos Antivirales/sangre , Fibrosis Quística/inmunología , Dependovirus/inmunología , Vectores Genéticos/inmunología , Inmunoglobulina G/sangre , Adulto , Linfocitos B/inmunología , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Distribución de Chi-Cuadrado , Citocinas/inmunología , Femenino , Humanos , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Linfocitos T/inmunologíaRESUMEN
Recombinant adeno-associated virus (rAAV) is a promising vector for therapy of retinal degenerative diseases. We evaluated the efficiency, cellular specificity, and safety of retinal cell transduction in nonhuman primates after subretinal delivery of an rAAV carrying a cDNA encoding green fluorescent protein (EGFP), rAAV. CMV.EGFP. The treatment results in efficient and stable EGFP expression lasting >1 year. Transgene expression in the neural retina is limited exclusively to rod photoreceptors. There is neither electroretinographic nor histologic evidence of photoreceptor toxicity. Despite significant serum antibody responses to the vector, subretinal readministration results in additional transduction events. The findings further characterize the retinal cell tropism of rAAV. They also support the development of studies aimed ultimately at treating inherited retinal degeneration by using rAAV-mediated gene therapy.
Asunto(s)
Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Transgenes , Animales , ADN Recombinante/metabolismo , Dependovirus , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Macaca mulatta , Microscopía FluorescenteRESUMEN
Triggering of CD4 coreceptors on both human and murine T cells can suppress TCR/CD3-induced secretion of IL-2. We show here that pretreatment of murine CD4+ T cells with the CD4-specific mAb YTS177 inhibits the CD3-mediated activation of the IL-2 promoter factors NF-AT and AP-1. Ligation of CD4 molecules on T cells leads to a transient stimulation of extracellular signal-regulated kinase (Erk) 2, but not c-Jun N-terminal kinase (JNK) activity. Pretreatment with anti-CD4 mAb impaired anti-CD3-induced Erk2 activation. Costimulation with anti-CD28 overcame the inhibitory effect of anti-CD4 Abs, by induction of JNK activation. The in vivo relevance of these studies was demonstrated by the observation that CD4+ T cells from BALB/c mice injected with nondepleting anti-CD4 mAb were inhibited in their ability to respond to OVA Ag-induced proliferation and IL-2 secretion. Interestingly, in vivo stimulation with anti-CD28 mAb restored IL-2 secretion. Furthermore, animals pretreated with anti-CD4 elicited enhanced IL-4 secretion induced by OVA and CD28. These observations suggest that CD4-specific Abs can inhibit T cell activation by interfering with signal 1 transduced through the TCR, but potentiate those delivered through the costimulatory molecule CD28. These studies have relevance to understanding the mechanism of tolerance induced by nondepleting anti-CD4 mAb used in animal models for allograft studies, autoimmune pathologies, and for immunosuppressive therapies in humans.