RESUMEN
CSF biomarkers, including total-tau, neurofilament light chain (NfL) and amyloid-ß, are increasingly being used to define and stage Alzheimer's disease. These biomarkers can be measured more quickly and less invasively in plasma and may provide important information for early diagnosis of Alzheimer's disease. We used stored plasma samples and clinical data obtained from 4444 non-demented participants in the Rotterdam study at baseline (between 2002 and 2005) and during follow-up until January 2016. Plasma concentrations of total-tau, NfL, amyloid-ß40 and amyloid-ß42 were measured using the Simoa NF-light® and N3PA assays. Associations between biomarker plasma levels and incident all-cause and Alzheimer's disease dementia during follow-up were assessed using Cox proportional-hazard regression models adjusted for age, sex, education, cardiovascular risk factors and APOE ε4 status. Moreover, biomarker plasma levels and rates of change over time of participants who developed Alzheimer's disease dementia during follow-up were compared with age and sex-matched dementia-free control subjects. During up to 14 years follow-up, 549 participants developed dementia, including 374 cases with Alzheimer's disease dementia. A log2 higher baseline amyloid-ß42 plasma level was associated with a lower risk of developing all-cause or Alzheimer's disease dementia, adjusted hazard ratio (HR) 0.61 [95% confidence interval (CI), 0.47-0.78; P < 0.0001] and 0.59 (95% CI, 0.43-0.79; P = 0.0006), respectively. Conversely, a log2 higher baseline plasma NfL level was associated with a higher risk of all-cause dementia [adjusted HR 1.59 (95% CI, 1.38-1.83); P < 0.0001] or Alzheimer's disease [adjusted HR 1.50 (95% CI, 1.26-1.78); P < 0.0001]. Combining the lowest quartile group of amyloid-ß42 with the highest of NfL resulted in a stronger association with all-cause dementia [adjusted HR 9.5 (95% CI, 2.3-40.4); P < 0.002] and with Alzheimer's disease [adjusted HR 15.7 (95% CI, 2.1-117.4); P < 0.0001], compared to the highest quartile group of amyloid-ß42 and lowest of NfL. Total-tau and amyloid-ß40 levels were not associated with all-cause or Alzheimer's disease dementia risk. Trajectory analyses of biomarkers revealed that mean NfL plasma levels increased 3.4 times faster in participants who developed Alzheimer's disease compared to those who remained dementia-free (P < 0.0001), plasma values for cases diverged from controls 9.6 years before Alzheimer's disease diagnosis. Amyloid-ß42 levels began to decrease in Alzheimer's disease cases a few years before diagnosis, although the decline did not reach significance compared to dementia-free participants. In conclusion, our study shows that low amyloid-ß42 and high NfL plasma levels are each independently and in combination strongly associated with risk of all-cause and Alzheimer's disease dementia. These data indicate that plasma NfL and amyloid-ß42 levels can be used to assess the risk of developing dementia in a non-demented population. Plasma NfL levels, although not specific, may also be useful in monitoring progression of Alzheimer's disease dementia.
Asunto(s)
Péptidos beta-Amiloides/sangre , Biomarcadores/sangre , Demencia/diagnóstico , Proteínas de Neurofilamentos/sangre , Proteínas tau/sangre , Anciano , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/diagnóstico , Estudios de Cohortes , Demencia/sangre , Diagnóstico Precoz , Femenino , Humanos , MasculinoRESUMEN
There is much debate in the pharmaceutical industry on how to translate the current guidelines on immunogenicity testing for biotherapeutics into a testing strategy that suits the specific requirements of individual drug candidates. In this paper, member companies from the European immunogenicity platform (EIP) present a consensus view on the essential requirements for immunogenicity testing of a biotherapeutic throughout the various phases of drug development, to ensure patient safety and to enable successful market entry. Our aim is to open the debate and provoke discussion on this important topic which is unique to biotherapeutic drug development. The scope of this paper is limited to aspects relevant to biotherapeutic drug development and does not include fundamental academic studies of immunogenicity. Here, we propose two pre-defined testing strategies for the detection and characterization of anti-drug antibody (ADA) responses where the different strategies are based on the phase of development for a biotherapeutic, a. without (category 1) and b. with (category 2) the expected potential to elicit ADA mediated severe clinical consequences. The harm of a potential ADA response determines which of the two testing strategies is adopted. Rather than replacing the overall risk assessment which is known to be challenging and multi-factorial, the testing strategy selection is a starting point for immunogenicity testing which adapts throughout drug development as more information becomes available. The scientific rationale on which the "case-by-case" approach advocated in white papers and guidance documents may be translated for each individual drug development program is provided and, underpins the recommendations made here.
Asunto(s)
Anticuerpos Neutralizantes/análisis , Terapia Biológica/efectos adversos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Industria Farmacéutica/tendencias , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/inmunología , Pruebas Inmunológicas/normas , Evaluación Preclínica de Medicamentos/normas , Europa (Continente) , Guías como Asunto , HumanosRESUMEN
BACKGROUND: As most vaccines exert their protective capacity by eliciting pathogen-specific antibodies, antibody assays assessing immunogenicity of vaccines in development should be well characterized. Part of the validation of immunogenicity assays for vaccines is the study of stability of antibodies in serum. Materials & methods: Stability of antibodies in human serum was assessed by circumsporozoite-binding IgG ELISA designed for assessing the immunogenicity of a malaria vaccine under development, adenovirus neutralization assay, designed to assess neutralizing antibodies against adenovirus and commercially available test kits for hepatitis A and B. RESULTS: Stability studies indicated stability of serum-binding IgG antibodies and serum-neutralizing antibodies in: long-term storage below -65°C and -20°C; short-term storage; multiple freeze/thaw rounds; during shipment; and during heat inactivation. CONCLUSION: RESULTS have shown the stability of both binding and functional polyclonal antibodies in human serum under stable storage and common usage circumstances.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Ensayo de Inmunoadsorción Enzimática , Vacunas/inmunología , Adenoviridae/inmunología , Congelación , Virus de la Hepatitis A/inmunología , Virus de la Hepatitis B/inmunología , Humanos , Estabilidad Proteica , Proteínas Protozoarias/inmunología , Análisis de Regresión , Temperatura , Factores de TiempoRESUMEN
All therapeutic proteins are potentially immunogenic. Antibodies formed against these drugs can decrease efficacy, leading to drastically increased therapeutic costs and in rare cases to serious and sometimes life threatening side-effects. Many efforts are therefore undertaken to develop therapeutic proteins with minimal immunogenicity. For this, immunogenicity prediction of candidate drugs during early drug development is essential. Several in silico, in vitro and in vivo models are used to predict immunogenicity of drug leads, to modify potentially immunogenic properties and to continue development of drug candidates with expected low immunogenicity. Despite the extensive use of these predictive models, their actual predictive value varies. Important reasons for this uncertainty are the limited/insufficient knowledge on the immune mechanisms underlying immunogenicity of therapeutic proteins, the fact that different predictive models explore different components of the immune system and the lack of an integrated clinical validation. In this review, we discuss the predictive models in use, summarize aspects of immunogenicity that these models predict and explore the merits and the limitations of each of the models.
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Evaluación Preclínica de Medicamentos/métodos , Proteínas/inmunología , Proteínas/uso terapéutico , Animales , Simulación por Computador , Humanos , Sistema Inmunológico/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Modelos BiológicosRESUMEN
Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Pruebas de Neutralización/métodos , Virus de la Rabia/inmunología , Anticuerpos Monoclonales/inmunología , Línea Celular , Humanos , Inmunoglobulinas/inmunología , Vacunas Antirrábicas/inmunologíaRESUMEN
Clinical development of vaccines requires a specific set of specialized assays to demonstrate the immunogenicity of the vaccine. Ideally, these assays should measure immune responses that correlate with protection against disease. Antibody responses usually correlates to protection for existing vaccines, but for vaccines currently in development it is not always clear which immune responses confer protection. Developing assays for new-generation vaccines usually requires working with cells, pathogens, antigens or assay controls that are not readily available, or are hazardous materials. Validation of these assays involves many challenges, and validation requirements are not yet fully specified in regulatory guidelines or White Papers. The different requirements for clinical vaccine assays and the related challenges in developing and validating these assays are described in this article. We provide our opinion on how to approach these challenges and how to apply the existing guidelines.
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Bioensayo , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Immunospot Ligado a Enzimas/métodos , Vacunas/análisis , Anticuerpos/análisis , Anticuerpos/inmunología , Formación de Anticuerpos/inmunología , Citocinas/análisis , Citocinas/inmunología , Humanos , Guías de Práctica Clínica como Asunto , Vacunas/inmunología , Estudios de Validación como AsuntoRESUMEN
Various pre-erythrocyte malaria vaccines are currently in clinical development, and among these is the adenovirus serotype 35-based circumsporozoite (CS) vaccine produced on PER.C6 cells. Although the immunological correlate of protection against malaria remains to be established, the CS antibody titer is a good marker for evaluation of candidate vaccines. Here we describe the validation of an anti-Plasmodium falciparum circumsporozoite antibody enzyme-linked immunosorbent assay (ELISA) based on the binding of antibodies to a peptide antigen mimicking the CS repeat region. The interassay variability was determined to be below a coefficient of variation (CV) of 15%, and sensitivity was sufficient to detect low antibody titers in subjects from endemic regions. Antibody titers were in agreement with total antibody responses to the whole CS protein. Due to its simplicity and high performance, the ELISA is an easy and rapid method for assessment of pre-erythrocyte malaria vaccines based on CS.
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Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos , Técnicas de Laboratorio Clínico/métodos , Vacunas contra la Malaria/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Adulto , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Malaria Falciparum/prevención & control , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
HLA-B57 has been shown to be associated with long-term asymptomatic HIV-1 infection. To investigate the biological mechanism by which the HLA-B57 allele could protect from HIV-1 disease, we studied both the number of CD8(+) T cells as well as CD8(+) T cell responsiveness directed to different HIV-1 Gag peptides presented by HLA-A2, -B8 or -B57. T cells specific for the HLA-B57 peptide KAFSPEVIPMF responded more readily and to a higher extend to antigenic stimulation in vitro than T cells specific for the HLA-A2 peptide SLYNTVATL or the HLA-B8 peptide EIYKRWII. This phenomenon was reproducible with T cells from individuals expressing HLA-B57 in combination with one or both of the other alleles and was persistent during long-term follow-up. Lower reactivity of A2- and B8-restricted T cells was not explained by mutations in the B8- or A2-restricted Gag-peptides. Moreover, no correlation between peptide mutation frequency and IFN-gamma production by the corresponding Gag-specific T cells was observed. In conclusion, functional differences were observed between T cells specific for HIV epitopes derived from the same protein presented by different HLA molecules. B57-restricted KAFSPEVIPMF-specific CD8(+) T cells have relatively high responsiveness, which could contribute to the protective effect of HLA-B57 in HIV infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Productos del Gen gag , Antígenos VIH , Infecciones por VIH/inmunología , Antígenos HLA-B/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/genética , Epítopos/genética , Productos del Gen gag/genética , Granzimas , Antígenos VIH/genética , VIH-1/genética , VIH-1/inmunología , Antígeno HLA-A2/metabolismo , Antígeno HLA-B8/metabolismo , Humanos , Técnicas In Vitro , Interferón gamma/biosíntesis , Glicoproteínas de Membrana/metabolismo , Mutación , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Studies in humans have provided evidence that CD8(+) T cells exhibit distinct phenotypical and functional properties dependent on virus specificity. It is not known how these T-cell phenotypes develop over the course of infection. Dynamics and properties of T cells specific for human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) in HIV infection were investigated in relation to viral load. In rapid progressors, HIV-specific CD8(+) T cells were less differentiated early in infection and did not develop a more differentiated phenotype. In slow progressors, perforin expression of HIV-specific CD8(+) T cells slightly increased over time. HIV and EBV loads were detectable in all individuals, while CMV load could not be detected. Thus, in individuals with progressive HIV infection, HIV-specific T cells are less differentiated already early in infection. This apparent block in differentiation may be partly caused by chronic viremia or lack of CD4(+) T-cell help.
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Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Citomegalovirus/fisiología , Progresión de la Enfermedad , Granzimas , VIH-1/inmunología , Herpesvirus Humano 4/fisiología , Humanos , Recuento de Linfocitos , Glicoproteínas de Membrana/metabolismo , Perforina , Fenotipo , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/metabolismo , Factores de Tiempo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Carga ViralRESUMEN
Substituting the coat proteins of adenoviral vector serotype 5 (Ad5) can alter vector tropism and circumvent vector neutralization. Here we report that an Ad5 vector carrying a part of the fiber molecule of human subgroup B adenovirus serotype 35 (Ad5.Fib35) transduces cultured human dendritic cells (DC) and circulating myeloid derived DC with approximately 10-fold greater efficiency than Ad5 in vitro. The improved DC transduction results in increased T-cell activation ex vivo. In vivo however, immunogenicity of the vectors in mice and non-human primates did not correlate with in vitro DC tropism. Ad5.Fib35 was less immunogenic in monkeys than Ad5, despite the improved primate DC tropism of Ad5.Fib35. In mice with high Ad5 vector-specific immunity, Ad5.Fib35 showed no significant difference in anti-insert immunity over Ad5 indicating that fiber exchange alone does not evade pre-existing Ad5 immunity. We thus conclude that, for ex vivo vaccination, Ad5.Fib35 shows promise as vector for loading of DC but is unable to circumvent anti-Ad5 immunity limiting its in vivo utility.
Asunto(s)
Adenoviridae/genética , Adenoviridae/inmunología , Células Dendríticas/inmunología , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Animales , Formación de Anticuerpos/inmunología , Proteínas de la Cápside/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Hemaglutininas/inmunología , Humanos , Interferón gamma/metabolismo , Macaca fascicularis , Sarampión/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Monocitos/inmunología , Pruebas de Neutralización , Distribución Tisular , Transducción Genética , Vacunación , Vacunas de ADN/genética , Vacunas de ADN/inmunologíaRESUMEN
The seroprevalence of adenovirus types 5 (Ad5) and 35 (Ad35) was investigated in patients at risk of AIDS. The seroprevalence of Ad5 was higher than Ad35 in HIV-infected patients from The Netherlands (60% versus 7%) and sub-Saharan Africa (90% versus 20%). The seroprevalence was similar among HIV-infected and uninfected individuals, and remained constant during progression to AIDS. Ad35 is less prone to neutralization than Ad5, encouraging the further development of Ad35 for vaccination against HIV.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/virología , Adenovirus Humanos/aislamiento & purificación , Anticuerpos Antivirales/sangre , Síndrome de Inmunodeficiencia Adquirida/sangre , Síndrome de Inmunodeficiencia Adquirida/inmunología , Adenovirus Humanos/inmunología , África del Sur del Sahara/epidemiología , Vectores Genéticos , Humanos , Países Bajos/epidemiología , Factores de Riesgo , Estudios SeroepidemiológicosRESUMEN
The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4(+) T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.
Asunto(s)
Infecciones por Adenoviridae/inmunología , Infecciones por Adenoviridae/prevención & control , Adenoviridae/genética , Adenoviridae/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología , Adenoviridae/clasificación , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Relación Dosis-Respuesta Inmunológica , Mapeo Epitopo/métodos , Epítopos de Linfocito T/sangre , Productos del Gen gag/administración & dosificación , Productos del Gen gag/sangre , Productos del Gen gag/inmunología , Vectores Genéticos , Inmunidad Activa , Esquemas de Inmunización , Inmunización Secundaria , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica/inmunología , Serotipificación , Virus de la Inmunodeficiencia de los Simios/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. Ad5-specific neutralizing antibodies (NAbs) are thought to contribute substantially to anti-Ad5 immunity, but the potential importance of Ad5-specific T lymphocytes in this setting has not been fully characterized. Here we assess the relative contributions of Ad5-specific humoral and cellular immune responses in blunting the immunogenicity of a rAd5-Env vaccine in mice. Adoptive transfer of Ad5-specific NAbs resulted in a dramatic abrogation of Env-specific immune responses following immunization with rAd5-Env. Interestingly, adoptive transfer of Ad5-specific CD8(+) T lymphocytes also resulted in a significant and durable suppression of rAd5-Env immunogenicity. These data demonstrate that NAbs and CD8(+) T lymphocytes both contribute to immunity to Ad5. Novel adenovirus vectors that are currently being developed to circumvent the problem of preexisting anti-Ad5 immunity should therefore be designed to evade both humoral and cellular Ad5-specific immune responses.
Asunto(s)
Infecciones por Adenovirus Humanos/inmunología , Adenovirus Humanos/inmunología , Anticuerpos Antivirales/sangre , Linfocitos T CD8-positivos/inmunología , Vectores Genéticos , Vacunas Virales/inmunología , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Traslado Adoptivo , Animales , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Serotipificación , Vacunas Virales/genéticaRESUMEN
Delivery of the full-length tumor antigen might be more successful in immunotherapy than single peptides and has the advantage that patients no longer need to be selected for their HLA type. In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by dendritic cells infected with an adenovirus (Ad) type 5 vector containing the fiber shaft and knob of human serotype Ad35 (Ad5F35 vector). Our data show induction of CD8(+) T cells specific for the known HLA-A(*)0201-binding CAMEL/NY-ESO-ORF2(1-11) epitope by DC infected with Ad5F35-CAMEL, but not by DC pulsed with the recombinant CAMEL protein. In one healthy donor, even CD8(+) T cells specific for a new HLA-B7-binding CAMEL/NY-ESO-ORF2(46-54) epitope were raised. In conclusion, the in vitro induction of CAMEL/NY-ESO-ORF2-specific CD8(+) T cells in healthy donors by DC infected with Ad5F35-CAMEL strongly supports further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the generation of tumor antigen-specific CD8(+) T cell responses in vivo.
Asunto(s)
Adenoviridae/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Vectores Genéticos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Animales , Antígenos de Superficie , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Cisteína Endopeptidasas/metabolismo , Mapeo Epitopo , Terapia Genética , Antígenos HLA-A/metabolismo , Antígeno HLA-B7/metabolismo , Humanos , Interleucinas/metabolismo , Complejos Multienzimáticos/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The presence of various levels of anti-adenovirus serotype 5 (Ad5)-neutralizing antibodies in humans is thought to contribute to the inconsistent clinical results obtained so far in diverse gene transfer and vaccination studies and might preclude universal dosing with recombinant Ad5. Prescreening of individuals eligible for Ad5 or alternative serotype treatment and subsequently tailoring the vector dose might aid in ensuring the consistency of clinical parameters. For this purpose, a qualified Ad neutralization assay is required. Here we have tested the different protocols used to date to determine anti-Ad neutralizing activity. Based on simplicity, speed, high throughput, sensitivity, and robustness, we propose a qualified assay in which Ad neutralization is monitored by luciferase reporter gene expression.
Asunto(s)
Adenoviridae/inmunología , Anticuerpos Antivirales/sangre , Luciferasas/genética , Adenoviridae/clasificación , Adenoviridae/genética , Adenoviridae/aislamiento & purificación , Adulto , Antígenos Virales/inmunología , Secuencia de Bases , Cartilla de ADN , Técnicas de Transferencia de Gen , Genes Reporteros , Genoma Viral , Humanos , Inmunoglobulina G/sangre , Luciferasas/análisis , Pruebas de Neutralización/métodos , Reacción en Cadena de la Polimerasa/métodos , Valores de ReferenciaRESUMEN
Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.
Asunto(s)
Adenovirus Humanos/inmunología , Adenovirus Humanos/fisiología , Vectores Genéticos , Replicación Viral , Proteínas E1B de Adenovirus/química , Proteínas E1B de Adenovirus/genética , Adenovirus Humanos/genética , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Células Cultivadas , Células Dendríticas/virología , Técnicas de Transferencia de Gen , Vectores Genéticos/administración & dosificación , Vectores Genéticos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Músculo Liso/citología , Músculo Liso/virología , Pruebas de Neutralización , Plásmidos , Membrana Sinovial/citología , Membrana Sinovial/virología , Vacunación , Ensamble de VirusRESUMEN
Despite readily detectable virus-specific CD8(+) T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. Recently developed insights into human T-cell differentiation have been used to study the phenotype of virus-specific T cells in HIV-infected individuals. Based on these results, we propose that failing immune control in human viral infection could be a result of impaired cytotoxic T-lymphocyte (CTL) maturation into fully differentiated effector T cells. Impaired maturation is not confined to HIV-specific CD8(+) T cells but could also be involved in failing immunity to Epstein-Barr virus and other viral infections. We postulate that CD27(-) effector CD8(+) T cells might be required for adequate control of chronic viral infection and prevention of disease development.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Virosis/inmunología , Linfocitos T CD8-positivos/patología , Diferenciación Celular , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/patología , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Humanos , Modelos Inmunológicos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Virosis/patologíaRESUMEN
OBJECTIVE: Despite readily detectable virus-specific CD8+ T cells in most HIV-infected patients, immune surveillance is eventually lost, leading to progression to AIDS. To investigate the underlying mechanism of this loss of immune control phenotypic analysis of HIV- and Epstein-Barr virus (EBV)-specific CD8+ T cells was performed. DESIGN: In three clinically distinct groups, long-term asymptomatics, progressors to opportunistic infections and progressors to EBV-associated non-Hodgkin lymphoma's (NHL), both number and phenotype of virus-specific CD8+ T cells was studied longitudinally. METHODS: The number of HIV- and EBV-specific T cells were determined using HLA-peptide tetrameric complexes. The phenotype of these virus-specific T cells was investigated by costaining with CD27 and CD45RO and thereby identifying specific subsets of CD8+ T cells. RESULTS: Individuals co-infected with HIV and EBV persistently had low numbers of HIV-specific CD27- T cells, in contrast to rising numbers of EBV-specific CD27- CD8+ T cells. However, HIV-infected individuals developing EBV-associated AIDS-related NHL had very low numbers of EBV-specific CD27- CD8+ T cells. Higher numbers of HIV-specific CD27- CD8+ T cells were associated with delayed disease progression. Virus-specific CD27- T cells, compared with CD27+ T cells showed elevated interferon-gamma production in response to viral peptides in vitro, indicative for strong effector function. CONCLUSIONS: Taken together, our data indicate that virus-specific CD27- T cells may be important effector T cells in controlling chronic viral infections in humans and that lack of differentiation into CD27- effector T cells may lead to progression of viral disease.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/fisiopatología , VIH-1/inmunología , Herpesvirus Humano 4/inmunología , Linfoma Relacionado con SIDA/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Adulto , Recuento de Linfocito CD4 , Linfocitos T CD8-positivos/fisiología , Progresión de la Enfermedad , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Linfoma Relacionado con SIDA/virología , Masculino , Persona de Mediana EdadRESUMEN
It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Sobrevivientes de VIH a Largo Plazo , VIH-1/inmunología , VIH-2/inmunología , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Subgrupos de Linfocitos T/inmunología , Adulto , Anciano , Fármacos Anti-VIH/uso terapéutico , Antígenos CD/análisis , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Estudios de Cohortes , Progresión de la Enfermedad , Exocitosis , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/fisiología , VIH-2/fisiología , Humanos , Integrina beta1/inmunología , Masculino , Persona de Mediana Edad , Perforina , Fenotipo , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfocitos T/inmunología , Carga Viral , Replicación ViralRESUMEN
Although CD8(+) T cells initially suppress human immunodeficiency virus (HIV) replication, cytotoxic T-cell precursor frequencies eventually decline and fail to prevent disease progression. In a longitudinal study including 16 individuals infected with HIV-1, we studied both the number and function of HIV-specific CD8(+) T cells by comparing HLA-peptide tetramer staining and peptide-induced interferon-gamma (IFN-gamma) production. Numbers of IFN-gamma-producing T cells declined during progression to acquired immunodeficiency syndrome (AIDS), whereas the number of tetramer+ T cells in many individuals persisted at high frequencies. Loss of IFN-gamma-producing T cells correlated with declining CD4(+) T-cell counts, consistent with the need of CD4(+) T-cell help in maintaining adequate CD8(+) T-cell function. These data indicate that the loss of HIV-specific CD8(+) T-cell activity is not due to physical depletion, but is mainly due to progressively impaired function of HIV-specific CD8(+) T cells.