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Plant platforms for the production of T1D candidate vaccines
No full textIl diabete di tipo 1 (T1D), definito anche diabete insulino-dipendente o diabete giovanile, è una malattia cronica nella quale si verifica una progressiva distruzione delle cellule β del pancreas, responsabili della produzione di insulina. La conseguente diminuzione di insulina provoca l’incremento della concentrazione di glucosio nel sangue e l’alterazione del metabolismo dei carboidrati, richiedendo una terapia sostitutiva continua che rende i pazienti totalmente dipendenti da iniezioni insuliniche.
L’incidenza in Europa di questa malattia nei bambini al di sotto dei 14 anni varia tra 11 e 25 nuovi casi all’anno ogni 100 000 bambini e i dati epidemiologici indicano che è destinata ad aumentare. La distruzione selettiva delle cellule β pancreatiche è un processo immuno-mediato legato all’inefficienza dei normali meccanismi responsabili del mantenimento della tolleranza verso gli antigeni autologhi, quali l’isoforma da 65 kDa della glutammato decarbossilasi (GAD65) e l’insulina; un ruolo autoantigenico nella malattia è stato inoltre ipotizzato per la proteina heat-shock 60 (HSP60), rilasciata dalle cellule β in seguito a stress.
Numerosi studi in modelli animali e in soggetti diabetici o a rischio di sviluppare la malattia, stanno testando la possibilità di prevenirne o bloccarne il decorso attraverso la somministrazione di autoantigeni o loro peptidi immunoreattivi, in grado di indurre un processo di tolleranza immunologica; tale strategia è detta immunoterapia antigene-specifica (ASI). I suoi effetti sono strettamente dipendenti dalla molecola scelta per il trattamento, dalla via di somministrazione e dallo stadio della patologia al momento dell’intervento. Questo approccio consente di eliminare o inattivare dal punto di vista funzionale le cellule T autoreattive in modo selettivo, ovvero senza compromettere la funzionalità del sistema immunitario.
Sulla base dei risultati positivi ottenuti nel modello animale (topo NOD - non obeso diabetico), test clinici nell’uomo miranti a valutare l’efficacia della somministrazione di insulina per via orale (NCT00419562) o intranasale (NCT00419562) sono attualmente in corso. Inoltre uno studio in fase II basato sulla somministrazione di GAD65 si sta svolgendo in Svezia, con lo scopo di testare l’efficacia di questa molecola nella prevenzione della malattia in soggetti pre-diabetici (NCT01122446).
Oltre che attraverso antigeni interi, la tolleranza immunologica può essere indotta attraverso la somministrazione di peptidi. In particolare è stato dimostrato che il trattamento di topi NOD con microgrammi di peptidi rappresentanti alcuni dei maggiori epitopi T derivanti dagli autoantiantigeni della malattia, può prevenire, ritardare o sopprimere in modo efficace il processo di patogenesi. Un peptide derivante da HSP60 (DiaPep277) è stato già testato in pazienti malati di T1D di recente insorgenza e due studi di fase III attualmente in corso, stanno valutando la sua efficacia nel preservare la funzionalità delle cellule β (NCT00615264 e NCT01103284).
Fra i maggiori svantaggi di questo tipo di terapia, vi è l’alto costo associato alla produzione delle molecole di interesse. Nella prospettiva di utilizzo dell’immunoterapia a livello più ampio come strategia di vaccinazione dei soggetti a rischio, è auspicabile lo sviluppo di sistemi che consentano la produzione delle molecole a costi minori rispetto ai sistemi attualmente utilizzati. Nel corso degli ultimi venti anni numerose evidenze sperimentali dimostrano che le piante possono essere utilizzate come biofabbriche per la produzione di molecole di interesse terapeutico, offrendo vantaggi in termini economici e di scalabilità.
L’obiettivo generale di questo progetto di dottorato, è la produzione di molecole utilizzabili come putativi vaccini del diabete autoimmune in diverse piattaforme vegetali, con lo scopo di sviluppare sistemi di produzione e purificazione delle molecole di interesse altamente efficienti ed economici.
In particolare, diverse forme della GAD65 sono state prodotte in piattaforme vegetali di vario tipo, dimostrando l’applicabilità di questo approccio per la produzione di tale proteina.
Inoltre, sfruttando il fatto che i virus vegetali possono essere utilizzati come supporto per l’esposizione di peptidi eterologhi, sono state prodotte particelle virali esponenti sulla loro superficie un epitopo della GAD65 umana, da testare nel modello animale per valutarne l’efficacia nella prevenzione della malattia.Type 1 diabetes (T1D) is an autoimmune disease characterized by the T-cell mediated destruction of insulin-secreting pancreatic β-cells, causing the need of life-long insulin therapy. The autoimmune response is targeted mainly against some proteins present on the β-cells, including the smaller isoform of glutamic acid decarboxylase (GAD65), insulin and the tyrosine phosphate-like IA-2/IA-2B antigen; an autoantigenic role of heat-shock protein 60 (HSP60) released from stressed β-cells has also been demonstrated. Clinical trials are currently testing the possibility of preventing the clinical onset of the disease or delaying the loss of -cell function using immunological strategies based on the induction of tolerance (Tisch & McDevitt, 1994). In this research field, many studies have focused on antigen-specific immunotherapy (ASI), which is based on the delivery of autoantigens or autoantigen-derived peptides at different disease stages and via different routes, resulting in self-tolerance and prevention, delay or suppression of the pathogenic process. ASI is an appealing strategy in autoimmune disease treatment because it selectively inactivates, eliminates, or functionally deviates autoreactive T cells while maintaining the function of the remainder of the immune system (Tisch et al., 1999). The effect is strongly dependent on dose, frequency, route of administration, use of adjuvants, interindividual variations and especially the molecule used for immunization (autoantigen chosen, use of the whole protein or single or mixed peptides) (Peakman et von Herrath, 2010).
Given the many potential advantages of this kind of therapy, several studies have been performed by the administration of whole T1D autoantigens or autoantigen peptides, aiming at preventing the disease or suppressing its pathogenic process. In particular, given the promising pre-clinical results obtained in T1D animal model (non obese diabetic -NOD- mouse), human trials based on oral (NCT00419562) and intranasal (NCT00336674) administration of whole insulin are currently underway. A phase II prevention trial based on the use of whole GAD65 (two injections of alum-formulated protein, one month apart) is also in progress in Sweden (NCT01122446). Alternatively, in the last decade many pre-clinical studies in the NOD mice have focused on the use of T1D epitopes from different autoantigens (mainly GAD65, insulin and proinsulin, HSP60). It was demonstrated that the administration of single or mixed T1D-epitopes in the range of μg (usually 40-600) can effectively prevent, delay or suppress the onset of the disease (Arai et al., 2010; Chen et al., 2009; Daniel et Wegmann, 1996; Eldor et al., 2009; Tian et al., 1996; Tisch et al., 1999; Ogino et al., 2000). A modified peptide from HSP60 (DiaPep277) was also tested in humans and two ongoing phase III trials in newly diagnosed T1D patients are assessing its efficacy in preserving β-cell function (NCT00615264 and NCT01103284).
The major disadvantage connected with this kind of therapy is the high cost associated with molecule production. In the perspective of ASI therapy for autoimmune treatment the production of immunoreactive molecules in cost-effective recombinant systems would be highly desirable.
For this purpose, plant-based systems represent useful platforms for the production of therapeutic proteins, offering advantages in terms of economy and scalability.
The aim of this Ph.D. project is the expression of different forms of T1D candidate vaccines in several plant expression platforms, in order to develop high-throughput and cost-effective systems for the production and purification of the target molecules, to use for antigen-specific immunotherapy of T1D.
Different forms of GAD65 (one of the major T1D autoantigen) were expressed in several plant-based platforms, demonstrating the feasibility of this approach to obtain high recombinant protein yields. Expression levels and molecular features of the protein obtained in the different plant systems are discussed.
Moreover, exploiting the fact that plant viruses can be used as scaffold to display peptides of interest on their surface, plant-viral particles displaying a GAD65 T1D epitope were produced. The efficacy of these molecules as novel candidate vaccine for the prevention of the disease will be tested in NOD mouse animal model
Edible plants for oral delivery of biopharmaceuticals
No full textMolecular farming is the use of plants for the production of high value recombinant proteins. Over the last 25 years, molecular farming has achieved the inexpensive, scalable and safe production of pharmaceutical proteins using a range of strategies. One of the most promising approaches is the use of edible plant organs expressing biopharmaceuticals for direct oral delivery. This approach has proven to be efficacious in several clinical vaccination and tolerance induction trials as well as multiple preclinical studies for disease prevention. The production of oral biopharmaceuticals in edible plant tissues could revolutionize the pharmaceutical industry by reducing the cost of production systems based on fermentation, and also eliminating expensive downstream purification, cold storage and transportation costs. This review considers the unique features that make plants ideal as platforms for the oral delivery of protein-based therapeutics and describes recent developments in the production of plant derived biopharmaceuticals for oral administration
Comparative Evaluation of Recombinant Protein Production in Different Biofactories: The Green Perspective
No full textIn recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms
Perspectives for autoimmune diabetes prevention using plants
No full textAutoimmune diabetes is the most frequent chronic disease of childhood and it is characterised by long-term and severe complications; different therapeutic approaches have been used in order to prevent and to suppress the disease at an experimental level but no one of them reached so far positive results in human trials. However, the results of clinical trials using autoantigen-based therapies suggested new insight for the research of a vaccine.
The 65 kDa isoform of human glutamic acid decarboxylase, one of the major autoantigen associated with the disease, is regarded as crucial for future studies on diabetes prevention and suppression.
GAD65 is actually produced using conventional expression systems (mainly based on yeast and baculovirus/insect cell) and the final product is characterised by high costs.
We exploited plants as a platform for the production of human GAD65 by using stable and transient expression and by engineering the protein at different degrees. Our results indicate that plants are a feasible and versatile system for the production of this recombinant protein. The results obtained in the perspective of new therapeutic approaches are described and discussed
A downstream process allowing the efficient isolation of a recombinant amphiphilic protein from tobacco leaves
No full textThe 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major autoantigen in autoimmune diabetes. The heterologous production of hGAD65 for diagnostic and therapeutic applications is hampered by low upstream productivity and the absence of a robust and efficient downstream process for product isolation. A tobacco-based platform has been developed for the production of an enzymatically-inactive form of the protein (hGAD65mut), but standard downstream processing strategies for plant-derived recombinant proteins cannot be used in this case because the product is amphiphilic. We therefore evaluated different extraction buffers and an aqueous micellar two-phase system (AMTPS) to optimize the isolation and purification of hGAD65mut from plants. We identified the extraction conditions offering the greatest selectivity for hGAD65mut over native tobacco proteins using a complex experimental design approach. Under our optimized conditions, the most efficient initial extraction and partial purification strategy achieved an overall hGAD65mut yield of 92.5% with a purification factor of 12.3 and a concentration factor of 23.8. The process also removed a significant quantity of phenols, which are major contaminants present in tobacco tissue. This is the first report describing the use of AMTPS for the partial purification of an amphiphilic recombinant protein from plant tissues and our findings could also provide a working model for the initial recovery and partial purification of hydrophobic recombinant proteins from transgenic tobacco plants
GFP-based expression of truncated GSD65mut forms in high-throughput platforms
No full textType 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the destruction of insulin-secreting pancreatic beta cells, causing the need of life-long insulin therapy. The 65 kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen involved in the disease development. Recent phase II clinical trials have shown that administration of alum-formulated GAD65 lead to a significant preservation of residual insulin secretion without serious adverse effects (Ludvigsson et al., 2008); large-scale confirmatory studies are underway in Europe and in the USA. Current systems for the production of large quantity of purified recombinant protein are very expensive and inadequate to provide enough GAD65 to meet the global demand. We have previously shown that GAD65 and a mutated catalytically-inactive form of the protein (GAD65mut) can be expressed in transgenic tobacco plants (Avesani et al., 2003; Avesani et al., 2010). GAD65mut accumulates 10-fold higher than GAD65 and retains the immunogenic properties required to treat T1DM (Avesani et al., 2010). In order to develop a system for the highefficient production and purification of GAD65, we engineered GAD65mut to various extents to obtain soluble forms of the molecule. It is well documented that GAD65 undergoes some posttranslational modifications in the N-terminal domain that result in a firmly membrane-anchored protein, which is only released by the use of detergents, thus complicating downstream processing of the protein. In the present work we described and discussed the solubility and accumulation levels of three N-truncated forms of GAD65mut in comparison with full-length GAD65mut and GAD65 in a plant-based and a yeast-based platform. The two platforms have been chosen for the high-throughput and fast expression of the molecules. Furthermore, in both the systems, GAD65- based forms have been C-terminal fused with GFP in order to easily determine the recombinant proteins sub-cellular localization
PVX a powerful tool for autoimmune disease diagnosis
No full textWe explored the use of Potato Virus X as a scaffold for the expression of an immunodominant peptide associated to Sjogren’s syndrome.
Sjögren's syndrome (SjS) is a chronic autoimmune disease in which the body's white cells destroy the exocrine glands, specifically the salivary and lacrimal glands, that produce saliva and tears, respectively.
Human lipocalin was recently identified as a primary autoantigen associated to the disease and the immunodominant peptide associated to the protein was discovered using the sera of SjS patients. Autoantibodies directed to the peptide allow to identify in the population patients whose sera was negative to anti nuclear antibodies and to the rheumatoid factor.
The lipocalin peptide was cloned into a PVX-based vector which mediates the peptide display on the virus coat protein, yielding Chimeric Virus Particles (CVPs). CVPs allow a high-density exposure of the peptide on the viral surface.
The CVPs were used for coating an ELISA plate giving an assay which reproducibility, stability and sensitive was compared to the use of the peptide alone
Expression of a mutated form of GAD65 in heterologous systems
No full textType 1 insulin-dependent diabetes mellitus (T1DM) which afflicts 0.2-0.3% of population is caused by autoimmune destruction of insulin-secreting beta cells. The young age of affected patients, the need for life-long insulin therapy and the high prevalence of late-onset complications make T1DM a major health problem. The smaller isoform of glutamic acid decarboxylase of 65 kDa (GAD65) is the major autoantigen in human T1DM and it has recently demonstarated that two injections of the molecule can give protection against this autoimmune disease. T1DM requires a primary prevention because the disease has a complex genetic basis, making difficult to identify in the population people at risk of developing it. Vaccination studies and subsequent vaccination treatment of a lot of people need large quantity of purified protein, but the current production systems are too much expensive and unable to provide enough GAD65 to meet global demand. We have previously shown that GAD65 can be expressed in transgenic tobacco plants but yields are disappointing. In order to improve its expression level we use different heterologous systems such as Nicotiana tabacum plants, E.coli inducible system and insect cells/Baculovirus to express two different forms of the recombinant human GAD65: the wild type form of the enzyme (hGAD65) and the mutated form with no catalytic activity (hGAD65mut), hypothesising that the enzymatic activity might interfere with its accumulation in heterologous systems. In previous studies it has been demonstrated in vitro the lack of the enzymatic activity for the hGAD65mut and we show that GAD65mut accumulates to higher levels in transgenic plants and in E.coli inducible system than its enzymatically active counterpart, indicating that the catalytic properties of GAD65 contribute to its poor yields. To demonstrated the absence of enzymatic activity of the mutated form of GAD65 (GAD65mut) also in the heterologous systems we perform an enzymatic assay in vivo,. The results of the assay and the difference among the expression levels obtained in the heterologous systems are discussed
Comparative analysis of different biofactories for the production of a major diabetes autoantigen.
No full textThe 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 μg/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 μg/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hand
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