In vitro and in vivo characterization of the RE-1 Silencing Transcription Factor (REST) activity under neuroinflammatory conditions

The ability to specifically target epigenetic and molecular mechanisms involved in neuronal development could be an alternative therapeutic strategy for neuroinflammatory/neurodegenerative disorders such as Multiple Sclerosis (MS). The transcriptional repressor RE1-Silencing Transcription Factor (REST) regulates neurogenesis and neuronal identity through cell-specific gene repression, allowing expression of its targets in mature neurons where REST is quiescent. REST dysregulation has been implicated in several neurodegenerative disorders, including Alzheimer and Huntington diseases, tumors of the nervous system, and epilepsy. We found that REST is overexpressed in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE), suggesting that its dysregulation might be an important factor in the pathogenesis of the disease. Starting from these observations, we have firstly analyzed the expression of REST target genes in EAE and characterized the cell-specificity of REST overexpression, investigating the differential contribution of neuronal and glial cell populations to REST upregulation. Moreover, in order to mimic the inflammatory EAE scenario, we have analyzed REST activity in primary neuron cultures treated with various pro-inflammatory cytokines. Altogether, this study provides the basis for understanding the molecular mechanisms of REST expression during brain inflammation and its implication in the subsequent neurodegenerative processes

Biological Engineering and Characterization of an HIV-1 Envelope-based Genomic Library

The numerous immune evasion strategies embodied in the HIV-1 envelope (Env) glycoprotein still represent a daunting challenge in the development of a safe and effective vaccine. Among the most defying of these evasive mechanisms is the tremendous genetic diversity associated with the immunodominance of highly variable regions of Env. Current vaccine design efforts aim to elicit broadly neutralizing antibodies (bnAbs), some of which are able to neutralize more than 90% of circulating HIV-1 strains. However, a complex co-evolution of virus and humoral immune response considerably impairs the development of bnAbs. To facilitate this process, novel Env immunogens as well as innovative selection technologies for their identification are required. The first part of this thesis concentrated on the biological engineering of an envelope-based sequential permutation library, specifically focusing on characterization and quality control of the library. Each residue in the external part of Env was substituted by 20 natural amino acids, thus creating a library of 658 sublibraries and approximately 13.000 variants. Simultaneously, the respective constitutively expressing cell lines were generated by stably transfecting every sublibrary into Flp-InTM T-Rex 293 cells with the with the goal to utilize the stable cell line (SCL) library in a mammalian cell display and cell sorting-based screening technology to identify Env variants with improved antigenicity. Comprehensive quality controls of plasmid DNA library and the respective stable cell line library were conducted to assess potential limitations. In-depth analysis of the pDNA revealed deletions of various lengths majorly in the Env region affecting about 48% of the library. However, these deletions occurred only in a small fraction within the sublibraries, thus the actual contaminations amounted to 6-18%, respectively, deeming the library still eligible to work on. Focusing on the CD4 binding site (CD4bs) of Env, diversity and amino acid distribution of the pDNA- and the stable cell line-library was analyzed by Next Generation Sequencing (NGS). Whereas pDNA exhibited an average diversity of 19 amino acids in the sublibraries with a nearly ideal distribution, stable cell lines demonstrated a considerable decrease in diversity by approximately 38%, as well as a highly uneven and random distribution of amino acids. It stood to reason that particularly insufficient integration of Env during the generation of stable cell lines contributed to this substantial loss of diversity, although many other factors (i.e. transfection efficiency of cells and reagents, transfection of individual Env variants, viability of cells, stage of cell cycle, etc.) could not be excluded as potential causes. To prevent the introduction of such a bias into the selection of beneficial Env variants, optimization of stable cell line generation is an essential prerequisite.The second project focused on the identification of improved Env candidates with favorable antigenicity from the stable cell line library. For this purpose, a mammalian cell display and cell sorting-based technology was applied that combines the benefits of i) single integration of Env into a distinct FRT site resulting in the linkage of genotype and phenotype, ii) inducible Env expression to prevent cytotoxicity effects, iii) translational coupling of Env and GFP enabling an indirect normalization for induced Env expression and iv) display on Hek293T cells, thus ensuring native folding and mammalian glycosylation. Using the CD4bs SCL library, twelve Env variants demonstrating increased (gain of binding, GoB) and decreased (loss of binding, LoB) affinity for the bnAb VRC01, respectively, were selected in a single round of cell sorting procedure. Strikingly, none of the detected GoB variants and merely three LoB candidates could be unequivocally validated by means of a FACS-based equilibrium titration. There were grounds for supposition, that limitations in the selection procedure as well as the low amino acid diversity in the SCLs contributed to the discrepancies in the dat

Intracellularly selected recombinant antibodies targeting \u3b2 Amyloid Oligomers

Targeting beta Amyloid (A\u3b2) peptide, in relation to the degenerative processes of Alzheimer Disease (AD), and studying the mechanisms of A\u3b2 misfolding, oligomerization and aggregation are currently two hot-topics in AD research. Both these aspects can be preferentially approached by the use of anti-A\u3b2 antibodies, in particular of those which are conformation specific, as demonstrated by in vitro studies and by in vivo immunotherapy. Here, we describe the generation of a large panel of anti-A\u3b2 scFvs recombinant antibodies, exploiting a novel in vivo, yeast two hybrid-based, approach developed in our laboratory: the \u201cIntracellular Antibody Capture Technology\u201d (IACT). In this way, we have selected and characterized a panel of 18 different anti-A\u3b2 scFvs, which show interesting features in vitro and in cells. IACT-selected anti-A\u3b2 scFvs are conformation specific versus A\u3b2 oligomers and show peculiar immunoreactivity pattern versus the in vivo produced A\u3b2 deposits in human AD brains. Moreover, our anti-A\u3b2 scFvs, being in vivo selected in the yeast cytoplasm, can be readily expressed as intracellular antibodies in mammalian cells, targeted to different cellular compartments, allowing new promising strategies to study the emerging role of intracellular A\u3b2 processing and oligomerization in AD pathology. The panel of IACT-selected scFvs under study represents a new tool in the survey of existing anti-A\u3b2 antibodies, and the unique characteristics of the selection strategy used for their isolation appear to be particularly suited for the selection of oligomeric specific anti-A\u3b2 antibodies. Furthermore, the recombinant nature of the antibodies makes them ideally suited for extracellular and for intracellular delivery, in vitro as well as in vivo

Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens.

Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4-0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design

Pre-clinical development of chimeric virus-like particles based HPV: HIV vaccines by using mammalian cell expression system: limitations and challenges

[eng] HPV and HIV-1 are important public health issues in some developing and industrialized countries, but an effective chimeric HPV:HIV preventive vaccine is still unachievable. We aimed to establish an alternative mammalian (293F) cell expression system combining with chromatographic purification methods to reach an appreciable expression level, purity and recovery rate of chimeric HPV:HIV VLPs. In this study, the chimeric HPV:HIV (L1:P18I10 and L1:T20) immunogens were designed and produced by using 293F expression system. The HPV:HIV VLPs were subsequently purified by a 3-step chromatographic method, including cation (CEC), size exclusion (SEC) and heparin affinity (H-AC) chromatography. Then, the in vitro stability, in vitro self assembly and morphology of purified HPV:HIV VLPs were confirmed by non-reducing SDS-PAGE, molecular mass assay, transmission electron microscopy (TEM) respectively. The sequential and conformational P18I10 and T20 peptides presented on chimeric HPV:HIV VLPs were further characterized by HIV-1 anti-V3 and anti-2F5 monoclonal antibodies in vitro by using Western blot and indirect ELISA. Finally, the immunogenicity of HPV:HIV VLPs were assessed in BALB/c mice model. Because the development and manufacturing of an immunogenic HPV:HIV vaccine is still unachievable, this study provided a baseline strategy that may be worthy to support the global efforts to develop novel chimeric VLP-based vaccines for controlling HPV and HIV-1 infection

Mitochondrial complex I activity in microglia sustains neuroinflammation

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3

Sector Monitoring and Evaluation Systems in the context of Changing Aid Modalities: The case of Niger‘s Health Sector

Within the context of the 2005 Paris Declaration (PD) and the 2008 Accra Agenda for Action (AAA) recipient countries have committed themselves to setting up transparent results-oriented reporting and assessment frameworks, while donors are expected to use these frameworks and to collaborate with recipients in order to strengthen recipient countries‘ systems. However, progress in this area is slow: only three out of 54 countries in the 2008 PD Survey had adequate results-oriented frameworks. Donors, from their side, are reluctant to rely on systems which are only partially developed, which simultaneously blocks the further elaboration and maturing of recipient systems. Progress at sector level is generally stronger and particularly within health and education sectors where, in the context of Sector Wide Approaches (SWAps), several initiatives have been taken to strengthen monitoring and evaluation (M&E) systems. Prior to strengthening an M&E system it is important to assess the strengths and weaknesses of the existing system, taking both M&E supply and demand sides into account. This working paper analyses the M&E system in the health sector of Niger and focuses on issues of policy, methodology, organisation (structure and linkages), capacity, participation of actors outside government and use of M&E outputs. The assessment of the M&E system in Niger‘s health sector shows a mixed picture of a partially developed system. When taking into account that Niger is one of the least developed countries in the world, with very weak scores on many health indicators, this outcome is more positive than expected. The very prominent role of donors might possibly be related to the scores obtained. The authors of this working document, however, argue that if M&E system strengthening is to a large extent pushed from the outside (donors) and not motivated through an internal M&E demand and supply side (both from within as well as outside government), it is likely that the outputs of the system as well as their use will be weak.

Development of new therapeutic approaches in mouse models of Amyotrophic Lateral Sclerosis

La sclérose latérale amyotrophique (SLA) est une pathologie neurodégénérative caractérisée par une perte progressive des neurones moteurs et par de l’atrophie musculaire menant à de la paralysie. Bien que plusieurs mécanismes pathologiques aient été élucidés, la SLA reste un mystère médical puisqu’il n’existe toujours pas de traitement efficace. Nous avons développé deux stratégies dans le but de traiter la SLA. La première stratégie fut de cibler la protéine SOD1 mal repliée (chapitre 2) et la deuxième consistait à traiter la neuroinflammation présente dans la maladie (chapitre 3). Dans le chapitre 2, nous avons tenté de diminuer le niveau de la protéine SOD1 mal repliée présent dans le système nerveux de souris transgéniques développant un phénotype de SLA. Pour ce faire, nous avons testé une nouvelle approche thérapeutique basée sur l’utilisation d’ « adeno-associated virus (AAV) ». Ce virus contient une séquence d’ADN qui encode pour un anticorps à chaîne unique variable (scFv). Cet anticorps est composé d’une des deux chaînes légères et lourdes de l’anticorps D3H5 ciblant de manière spécifique la protéine SOD1 mal repliée. Une injection intra-thécale unique de l’AAV encodant l’anticorps à chaîne unique dans des souris SOD1G93A repousse le début de la maladie et augmente la survie des souris de 28%. Nous avons démontré que la Withaferin A (WA), un inhibiteur du facteur NF-κB, diminuait le phénotype neurologique retrouvé chez le modèle de souris transgénique TDP-43. Donc, nous avons testé la Withaferin A sur deux autres lignées de souris transgéniques exprimant des mutations dans la protéine SOD1, soit la lignée de souris SOD1G93A et la lignée de souris SOD1G37R. L’effet bénéfique de la WA chez les souris SOD1G93A était accompagné d’un soulagement de la neuroinflammation, d’une diminution du niveau de protéine SOD1 mal repliée dans la moelle épinière et d’une baisse de la mortalité des neurones moteurs. En considérant ces résultats, l’utilisation d’AAV encodant un anticorps à chaîne unique contre la protéine SOD1 mal repliée ainsi que l’utilisation de Withaferin A devrait toutes les deux être considérées comme des approches pour traiter la SLA.Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease associated with motor neuron degeneration, muscle atrophy and paralysis. Although numerous pathological mechanisms have been elucidated, ALS still remains a medical mystery in the absence of any effective therapy. Riluzole is the only therapeutic drug approved for ALS with regard to prolonging survival. Here, we have developed two strategies for treatment of ALS, first targeting the misfolded SOD1 (chapter 2) and other targeting neuroinflammation (chapter 3). In chapter 2, we aimed to reduce the level of misfolded SOD1 species in the nervous system. We tested a novel therapeutic approach based on adeno-associated virus (AAV)-mediated tonic expression of a DNA construct encoding a secretable single chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the adeno-associated virus encoding the single chain antibody in SOD1G93A mice delayed disease onset and extended the life span by up to 28%, in direct correlation with scFv titers in the spinal cord. Our second treatment strategy which is aimed to target neuroinflammation is based on previous reports from our lab where it has been shown that Withaferin A (WA), an inhibitor of NF-κB activity was efficient in reducing disease phenotype in TDP-43 transgenic mouse model of ALS. We tested WA in mice from two transgenic lines expressing different ALS-linked SOD1 mutations, SOD1G93A and SOD1G37R. The beneficial effects of WA in SOD1G93A mice model was accompanied by alleviation of neuroinflammation, decrease in level of misfolded SOD1 species in spinal cord, a reduction in loss of motor neurons, resulting in delayed disease progression and mortality. Based on these evidences, AAV encoding a secretable scFv against misfolded SOD1 and WA should be considered as a potential treatment for ALS

Neuronal pathology in targeted cortical experimental autoimmune encephalomyelitis and multiple sclerosis

In den letzten Jahren ist zunehmend deutlich geworden, dass die Multiple Sklerose (MS) nicht nur eine Erkrankung der weißen Substanz des zentralen Nervensystems ist, sondern auch häufig und beträchtlich die graue Substanz in allen klinischen Verlaufsformen betrifft. Besonders die kortikale Pathologie mit entmarkten Läsionen wurde durch verbesserte immunhistochemische Färbetechniken und neuen magnetresonanztomographischen Verfahren ausführlicher untersucht. MS-Patienten leiden klinisch oft an körperlichen Beeinträchtigungen und neuropsychologischen Defiziten, welche die Lebensqualität beeinflussen. Diese Symptome wurden mit Läsionen in der grauen Substanz assoziiert. Mechanismen, die zu dieser Pathologie führen, müssen daher aufgeklärt werden um vorbeugende oder akute Behandlungen entwickeln zu können. Zur pathologischen Untersuchung der grauen Substanz werden angemessene Tiermodelle benötigt, welche die humane kortikale Pathologie wiederspiegeln. Das am häufigsten verwendete Tiermodell in MS-Studien ist die Experimentelle Autoimmune Enzephalomyelitis (EAE), die in ihrem ‘konventionellen’ Immunisierungsprotokoll nur selten den zerebralen Kortex betrifft. Ein EAE-Modell mit Einbezug des Kortex, das MS-Läsionen nachahmt, wurde in Ratten beschrieben. Hierzu wurden proinflammatorische Zytokine in eine vorbestimmte kortikale Region injiziert. Da spezifisch genveränderte Rattenstämme fehlen um die Mechanismen der Pathologie in der grauen Substanz zu untersuchen ist es notwendig das Tiermodell in Mäusen zu entwickelen. Das Ziel dieses Projekts war die Entwicklung eines kortikalen EAE-Mausmodells sowie dessen histopathologische Charakterisierung. Desweiteren wurde kortikales Gehirnmaterial von MS-Patienten im späten Krankheitsstadium auf dendritische Patholgie untersucht. Die kortikale EAE wurde in Myelin Oligodendrozyten Glykoprotein (MOG)-immunisierten BiozziABH (hohe Antikörper) und F1 Nachkommen, die aus BiozziABH und Mäusen mit einem C57BL6/J-Hintergrund generiert worden sind, durch die intrakortikale Injektion von TNF-α und IFN-γ induziert. Histologische Untersuchungen zeigten eine ausgedehnte subpiale Entmarkung und Entzündung im Kortex drei Tage nach der Zytokininjektion in der betroffenen Hirnhälfte. Die Entzündung ging innerhalb von drei Wochen fast vollständig zurück und entmarkte Regionen wiesen teilweise eine Remyelinisierung auf. Axone blieben in läsionalen Regionen erhalten und neuronaler Verlust wurde im Kortex nicht beobachtet. Desweiteren wurde eine Methode etabliert, die es erlaubt detailliert dendritische Pathologien in der Maus zu untersuchen. Kortex-enthaltenes Autopsiematerial von progressiven MS-Patienten mit langandauerndem Krankheitsverlauf zeigte einen Verlust von dendritischen Dornfortsätzen (Spines) in Neurone, die in den unteren korikalen Layern sowohl in chronisch entmarkten Läsionen als auch im umliegenden normal erscheinendem Gewebe der grauen Substanz lokalisiert waren. Im vorliegenden Projekt wurde ein kortikales EAE-Mausmodell entwickelt, das die humane MS-Pathologie der grauen Substanz in frühen Krankheitsstadien wiederspiegelt. Dieses Modell ist für Untersuchungen früher Mechanismen im entmarkten Kortex und für die Erprobung therapeutischer Behandlungen wie die Erhöhung der Remyelinisierung nützlich. Darüberhinaus wurde ein ausgedehnter Verlust dendritischer Dornfortsätze im zerebralen Kortex in chronischen MS-Patienten gezeigt, der auf oft beobachtete neuropsychologische Defizite zurückgeführt werden könnte

Development of oligomer-specific antibodies against tau protein and testing of therapeutic potential in a cell model of tau pathology

Tau, a microtubule associated protein, forms abnormal aggregates in many neurodegenerative diseases such as Alzheimer disease (AD). There is an urgent need for disease-modifying therapies of AD and related tauopathies. Inhibiting the aggregation of tau and the accumulation of neurofibrillary tangles (NFTs) could be helpful in combating tau pathology. Recent studies show that tau induced toxicity is mainly due to the presence of oligomers of tau rather than the monomers and fibrillar aggregates (Kaniyappan et al., 2017, Flach et al., 2012, Lasagna-Reeves et al., 2010). To combat the toxicity of tau oligomers we developed antibodies against the purified low-n tau oligomers (dimers to hexamers) of TauRDΔK, the strongly aggregating repeat domain of tau. Monoclonal antibodies were tested by various biochemical and biophysical methods for their specificity to bind to the toxic oligomers. Some antibodies show specificity to aggregates of tau while others detect all forms of tau. Antibodies 2B10 and 6H1, described as representative examples, bind to tau oligomers with high specificity as judged by dotblot, dynamic light scattering (DLS) and immunofluorescence analysis. As these antibodies are dependent on tau conformations, they appear non-specific in denaturing methods like western blotting. 2B10 and 6H1 antibodies are able to inhibit the tau aggregation up to ~90% in vitro (TauRDΔK, hTauP301L), as judged by the Thioflavin S fluorescence assay which is sensitive to ß-structure. In the presence of antibodies tau protein forms only up to low-n oligomers as judged by light scattering and atomic force microscopy (AFM). The choice of the pH of the column elution buffer of the antibodies plays a key role in determining the activity of the antibodies, as antibodies eluted at low pH have a higher activity compared to the same antibodies eluted at high pH. The ability of antibodies to inhibit the aggregation of tau was tested in an N2a cell model of tau pathology which expresses the pro-aggregant tau repeat domain TauRDΔK. Antibodies were added to the extracellular medium, without or with protein transfection reagent (Xfect) which stimulates cellular uptake. In this assay, 2B10 antibody failed to inhibit tau aggregation (ThS signal) and failed to prevent aggregation induced apoptosis (Annexin V signal). By contrast, in the split-luciferase complementation assay the antibody 2B10, applied extracellularly, was able to prevent the dimerization/oligomerization of tau. Surprisingly this antibody has only a relatively low affinity to tau but is still very active in inhibiting tau aggregation in vitro. Antibodies added extracellularly were taken up by the cells and sorted into lysosomes. Their inhibitory effect can be explained by the fact that the internalized antibody recruits the toxic tau protein or oligomers to the lysosomes for degradation. In summary, a subset of antibodies raised against the purified low-n oligomers of TauRDΔK are able to inhibit tau aggregation both in vitro and in a cell model of tau pathology