RESUMO
Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aß) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aß represents a major constituent of the amyloid plaque. The data show that pE3Aß formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE3Aß accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE3Aß3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aß and 103-104 against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease.
Assuntos
Doença de Alzheimer , Vacinas Anticâncer , Camundongos , Animais , Doença de Alzheimer/prevenção & controle , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Ácido Pirrolidonocarboxílico , Imunoterapia , Placa Amiloide/patologia , Encéfalo/metabolismo , Camundongos Transgênicos , Modelos Animais de DoençasRESUMO
The current vaccine development strategies for the COVID-19 pandemic utilize whole inactive or attenuated viruses, virus-like particles, recombinant proteins, and antigen-coding DNA and mRNA with various delivery strategies. While highly effective, these vaccine development strategies are time-consuming and often do not provide reliable protection for immunocompromised individuals, young children, and pregnant women. Here, we propose a novel modular vaccine platform to address these shortcomings using chemically synthesized peptides identified based on the validated bioinformatic data about the target. The vaccine is based on the rational design of an immunogen containing two defined B-cell epitopes from the spike glycoprotein of SARS-CoV-2 and the universal T-helper epitope PADRE. The epitopes were conjugated to short DNA probes and combined with a complementary scaffold strand, resulting in sequence-specific self-assembly. The immunogens were then formulated by conjugation to gold nanoparticles by three methods or by co-crystallization with epsilon inulin. BALB/C mice were immunized with each formulation, and the IgG immune responses and virus neutralizing titers were compared. The results demonstrate that this assembly is immunogenic and generates neutralizing antibodies against wildtype SARS-CoV-2 and the Delta variant.
Assuntos
COVID-19 , Nanopartículas Metálicas , Complicações Infecciosas na Gravidez , Vacinas Virais , Gravidez , Camundongos , Animais , Feminino , Humanos , SARS-CoV-2 , Vacinas contra COVID-19 , Glicoproteína da Espícula de Coronavírus/química , Pandemias/prevenção & controle , COVID-19/prevenção & controle , Ouro , Camundongos Endogâmicos BALB C , Anticorpos Neutralizantes , Epitopos de Linfócito B/química , Anticorpos AntiviraisRESUMO
The interplay between the mechanical properties of double-stranded and single-stranded DNA is a phenomenon that contributes to various genetic processes in which both types of DNA structures coexist. Highly stiff DNA duplexes can stretch single-stranded DNA (ssDNA) segments between the duplexes in a topologically constrained domain. To evaluate such an effect, we designed short DNA nanorings in which a DNA duplex with 160 bp is connected by a 30 nt single-stranded DNA segment. The stretching effect of the duplex in such a DNA construct can lead to the elongation of ssDNA, and this effect can be measured directly using atomic force microscopy (AFM) imaging. In AFM images of the nanorings, the ssDNA regions were identified, and the end-to-end distance of ssDNA was measured. The data revealed a stretching of the ssDNA segment with a median end-to-end distance which was 16% higher compared with the control. These data are in line with theoretical estimates of the stretching of ssDNA by the rigid DNA duplex holding the ssDNA segment within the nanoring construct. Time-lapse AFM data revealed substantial dynamics of the DNA rings, allowing for the formation of transient crossed nanoring formations with end-to-end distances as much as 30% larger than those of the longer-lived morphologies. The generated nanorings are an attractive model system for investigation of the effects of mechanical stretching of ssDNA on its biochemical properties, including interaction with proteins.
Assuntos
DNA de Cadeia Simples , DNA , Estresse Mecânico , DNA/química , Microscopia de Força Atômica/métodos , Proteínas de Ligação a DNA/metabolismoRESUMO
Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by the aberrant accumulation of intracytoplasmic misfolded and aggregated α-synuclein (α-Syn), resulting in neurodegeneration associated with inflammation. The propagation of α-Syn aggregates from cell to cell is implicated in the spreading of pathological α-Syn in the brain and disease progression. We and others demonstrated that antibodies generated after active and passive vaccinations could inhibit the propagation of pathological α-Syn in the extracellular space and prevent/inhibit disease/s in the relevant animal models. We recently tested the immunogenicity and efficacy of four DNA vaccines on the basis of the universal MultiTEP platform technology in the DLB/PD mouse model. The antibodies generated by these vaccines efficiently reduced/inhibited the accumulation of pathological α-Syn in the different brain regions and improved the motor deficit of immunized female mice. The most immunogenic and preclinically effective vaccine, PV-1950D, targeting three B-cell epitopes of pathological α-Syn simultaneously, has been selected for future IND-enabling studies. However, to ensure therapeutically potent concentrations of α-Syn antibodies in the periphery of the vaccinated elderly, we developed a recombinant protein-based MultiTEP vaccine, PV-1950R/A, and tested its immunogenicity in young and aged D-line mice. Antibody responses induced by immunizations with the PV-1950R/A vaccine and its homologous DNA counterpart, PV-1950D, in a mouse model of PD/DLB have been compared.
Assuntos
Doença por Corpos de Lewy , Doença de Parkinson , Vacinas de DNA , Animais , Anticorpos , Modelos Animais de Doenças , Epitopos de Linfócito B , Feminino , Doença por Corpos de Lewy/metabolismo , Camundongos , Doença de Parkinson/metabolismo , Proteínas Recombinantes , alfa-Sinucleína/metabolismoRESUMO
The DNA vaccine, AV-1959D, targeting N-terminal epitope of Aß peptide, has been proven immunogenic in mice, rabbits, and non-human primates, while its therapeutic efficacy has been shown in mouse models of Alzheimer's disease (AD). Here we report for the first time on IND-enabling biodistribution and safety/toxicology studies of cGMP-grade AV-1959D vaccine in the Tg2576 mouse model of AD. We also tested acute neuropathology safety profiles of AV-1959D in another AD disease model, Tg-SwDI mice with established vascular and parenchymal Aß pathology in a pre-clinical translational study. Biodistribution studies two days after the injection demonstrated high copy numbers of AV-1959D plasmid after single immunization of Tg2576 mice at the injection sites but not in the tissues of distant organs. Plasmids persisted at the injection sites of some mice 60 days after vaccination. In Tg2576 mice with established amyloid pathology, we did not observe short- or long-term toxicities after multiple immunizations with three doses of AV-1959D. Assessment of the repeated dose acute safety of AV-1959D in cerebral amyloid angiopathy (CAA) prone Tg-SwDI mice did not reveal any immunotherapy-induced vasogenic edema detected by magnetic resonance imaging (MRI) or increased microhemorrhages. Multiple immunizations of Tg-SwDI mice with AV-1959D did not induce T and B cell infiltration, glial activation, vascular deposition of Aß, or neuronal degeneration (necrosis and apoptosis) greater than that in the control group determined by immunohistochemistry of brain tissues. Taken together, the safety data from two different mouse models of AD substantiate a favorable safety profile of the cGMP grade AV-1959D vaccine supporting its progression to first-in-human clinical trials.
Assuntos
Vacinas contra Alzheimer/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Doença de Alzheimer/imunologia , Doença de Alzheimer/prevenção & controle , Peptídeos beta-Amiloides/metabolismo , Animais , Formação de Anticorpos , Angiopatia Amiloide Cerebral/imunologia , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismoRESUMO
The self-assembly of amyloid ß (Aß) proteins into oligomers is the major pathogenic event leading to Alzheimer's disease (AD). Typical in vitro experiments require high protein concentrations, whereas the physiological concentration of Aß is in the picomolar to low nanomolar range. This complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here, we demonstrate that Aß42 self-assembles into aggregates on membrane bilayers at low nanomolar concentrations - a pathway in which the membrane plays the role of a catalyst. Additionally, physiological ionic conditions (150 mM NaCl) significantly enhance on-membrane aggregation, leading to the rapid formation of oligomers. The self-assembly process is reversible, so assembled aggregates can dissociate from the membrane surface into the bulk solution to further participate in the aggregation process. Molecular dynamics simulations demonstrate that the transient membrane-Aß interaction dramatically changes the protein conformation, facilitating the assembly of dimers. The results indicate peptide-membrane interaction is the critical step towards oligomer formation at physiologically low protein concentrations.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Doença de Alzheimer/genética , Humanos , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Within the last two decades, SARS-CoV-2 was the third zoonotic severe acute respiratory betacoronavirus (sarbecovirus) to infect humans, following SARS and MERS. The disruptions caused by the pandemic underscore the need for a universal vaccine against respiratory betacoronaviruses. Our group previously developed the universal platform for vaccine development, MultiTEP, which has been utilized in this study to generate a range of SARS-CoV-2 epitope vaccine candidates. We prepared and characterized 18 vaccines incorporating small peptide fragments from SARS-CoV-2 Spike protein fused with the MultiTEP sequence using overlapping PCR. Wild-type mice were immunized intramuscularly with the immunogen formulated in AdvaxCpG adjuvant. Serum antibodies were detected by ELISA, surrogate neutralization, and pseudovirus neutralization assays. Finally, the most promising vaccine candidate was administered to three non-human primates. All vaccines generated high titers of spike-binding IgG antibodies. However, only three vaccines generated antibodies that blocked RBD binding to the ACE2 receptor in a surrogate virus neutralization assay. However, none of the vaccines induced antibodies able to neutralize pseudotype viruses, including after the administration of the lead vaccine to NHPs. MultiTEP-based COVID-19 vaccines elicited robust, IgG-binding responses against the Spike protein in mice and non-human primates, but these antibodies were not neutralizing, underscoring the need to refine this approach further.
RESUMO
The current vaccine development strategies for the COVID-19 pandemic utilize whole inactive or attenuated viruses, virus-like particles, recombinant proteins, and antigen-coding DNA and mRNA with various delivery strategies. While highly effective, these vaccine development strategies are time-consuming and often do not provide reliable protection for immunocompromised individuals, young children, and pregnant women. Here, we propose a novel modular vaccine platform to address these shortcomings using chemically synthesized peptides and identified based on the validated bioinformatic data about the target. The vaccine is based on the rational design of an immunogen containing two defined B-cell epitopes from the spike protein of SARS-Co-V2 and a universal T-helper epitope PADRE assembled on the DNA scaffold. The results demonstrate that this assembly is immunogenic and generates neutralizing antibodies against SARS-CoV-2 wild type and its variants of concerns (VOC). This newly designed peptide nanoarray scaffold vaccine is useful in controlling virus transmission in immunocompromised individuals, as well as individuals who are prone to vaccine-induced adverse reactions. Given that the immunogen is modular, epitopes or immunomodulatory ligands can be easily introduced in order to tailor the vaccine to the recipient. This also allows the already developed vaccine to be modified rapidly according to the identified mutations of the virus.
RESUMO
Pathological forms of Tau protein are directly associated with neurodegeneration and correlate with Alzheimer's Disease (AD) symptoms, progression, and severity. Previously, using various mouse models of Tauopathies and AD, we have demonstrated the immunogenicity and efficacy of the MultiTEP-based adjuvanted vaccine targeting the phosphatase activating domain (PAD) of Tau, AV-1980R/A. Here, we analyzed its immunogenicity in non-human primates (NHP), the closest phylogenic relatives to humans with a similar immune system, to initiate the transition of this vaccine into clinical trials. We have demonstrated that AV-1980R/A is highly immunogenic in these NHPs, activating a broad but unique to each monkey repertoire of MultiTEP-specific T helper (Th) cells that, in turn, activate B cells specific to PAD. The resulting anti-PAD IgG antibodies recognize pathological Tau tangles and Tau-positive neuritis in AD case brain sections with no staining in control non-AD cases. These published data and efficacy results support the AV-1980R/A vaccine progression to first-in-human clinical trials.
RESUMO
Accumulation of misfolded proteins such as amyloid-ß (Aß), tau, and α-synuclein (α-Syn) in the brain leads to synaptic dysfunction, neuronal damage, and the onset of relevant neurodegenerative disorder/s. Dementia with Lewy bodies (DLB) and Parkinson's disease (PD) are characterized by the aberrant accumulation of α-Syn intracytoplasmic Lewy body inclusions and dystrophic Lewy neurites resulting in neurodegeneration associated with inflammation. Cell to cell propagation of α-Syn aggregates is implicated in the progression of PD/DLB, and high concentrations of anti-α-Syn antibodies could inhibit/reduce the spreading of this pathological molecule in the brain. To ensure sufficient therapeutic concentrations of anti-α-Syn antibodies in the periphery and CNS, we developed four α-Syn DNA vaccines based on the universal MultiTEP platform technology designed especially for the elderly with immunosenescence. Here, we are reporting on the efficacy and immunogenicity of these vaccines targeting three B-cell epitopes of hα-Syn aa85-99 (PV-1947D), aa109-126 (PV-1948D), aa126-140 (PV-1949D) separately or simultaneously (PV-1950D) in a mouse model of synucleinopathies mimicking PD/DLB. All vaccines induced high titers of antibodies specific to hα-Syn that significantly reduced PD/DLB-like pathology in hα-Syn D line mice. The most significant reduction of the total and protein kinase resistant hα-Syn, as well as neurodegeneration, were observed in various brain regions of mice vaccinated with PV-1949D and PV-1950D in a sex-dependent manner. Based on these preclinical data, we selected the PV-1950D vaccine for future IND enabling preclinical studies and clinical development.
RESUMO
The formation of amyloid ß (1-42) (Aß42) oligomers is considered to be a critical step in the development of Alzheimer's disease (AD). However, the mechanism underlying this process at physiologically low concentrations of Aß42 remains unclear. We have previously shown that oligomers assemble at such low Aß42 monomer concentrations in vitro on phospholipid membranes. We hypothesized that membrane composition is the factor controlling the aggregation process. Accumulation of cholesterol in membranes is associated with AD development, suggesting that insertion of cholesterol into membranes may initiate the Aß42 aggregation, regardless of a low monomer concentration. We used atomic force microscopy (AFM) to test the hypothesis and directly visualize the aggregation process of Aß42 on the surface of a lipid bilayer depending on the cholesterol presence. Time-lapse AFM imaging unambiguously demonstrates that cholesterol in the lipid bilayer significantly enhances the aggregation process of Aß42 at nanomolar monomer concentration. Quantitative analysis of the AFM data shows that both the number of Aß42 oligomers and their sizes grow when cholesterol is present. Importantly, the aggregation process is dynamic, so the aggregates assembled on the membrane can dissociate from the bilayer surface into the bulk solution. Computational modeling demonstrated that the lipid bilayer containing cholesterol had an elevated affinity to Aß42. Moreover, monomers adopted the aggregation-prone conformations present in amyloid fibrils. The results lead to the model for the on-surface aggregation process in which the self-assembly of Aß oligomers is controlled by the lipid composition of cellular membranes.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Amiloide , Colesterol , Humanos , Bicamadas Lipídicas , Fragmentos de PeptídeosRESUMO
The importance of cell surfaces in the self-assembly of proteins is widely accepted. One biologically significant event is the assembly of amyloidogenic proteins into aggregates, which leads to neurodegenerative disorders like Alzheimer's and Parkinson's diseases. The interaction of amyloidogenic proteins with cellular membranes appears to dramatically facilitate the aggregation process. Recent findings indicate that, in the presence of surfaces, aggregation occurs at physiologically low concentrations, suggesting that interaction with surfaces plays a critical role in the disease-prone aggregation process. However, the molecular mechanisms behind the on-surface aggregation process remain unclear. Here, we provide a theoretical model that offers a molecular explanation. According to this model, monomers transiently immobilized to surfaces increase the local monomer protein concentration and thus work as nuclei to dramatically accelerate the entire aggregation process. This physical-chemical theory was verified by experimental studies, using mica surfaces, to examine the aggregation kinetics of amyloidogenic α-synuclein protein and non-amyloidogenic cytosine deaminase APOBEC3G.
Assuntos
Desaminase APOBEC-3G/metabolismo , Proteínas Amiloidogênicas/metabolismo , Multimerização Proteica , alfa-Sinucleína/metabolismo , Desaminase APOBEC-3G/química , Silicatos de Alumínio/química , Proteínas Amiloidogênicas/química , Membrana Celular/metabolismo , Cinética , Microscopia de Força Atômica , alfa-Sinucleína/químicaRESUMO
The spontaneous self-assembly of α-synuclein (α-syn) into aggregates of different morphologies is associated with the development of Parkinson's disease. However, the mechanism behind the spontaneous assembly remains elusive. The current study shows a novel effect of phospholipid bilayers on the assembly of the α-syn aggregates. Using time-lapse atomic force microscopy, it was discovered that α-syn assembles into aggregates on bilayer surfaces, even at the nanomolar concentration range. The efficiency of the aggregation process depends on the membrane composition, with the greatest efficiency observed for of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS). Importantly, assembled aggregates can dissociate from the surface, suggesting that on-surface aggregation is a mechanism by which pathological aggregates may be produced. Computational modeling revealed that dimers of α-syn assembled rapidly, through the membrane-bound monomer on POPS bilayer, due to an aggregation-prone orientation of α-syn. Interaction of α-syn with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) leads to a binding mode that does not induce a fast assembly of the dimer. Based on these findings, we propose a model in which the interaction of α-syn with membranes plays a critical role initiating the formation of α-syn aggregates and the overall aggregation process.
Assuntos
Simulação por Computador , Bicamadas Lipídicas/química , Modelos Químicos , Agregados Proteicos , alfa-Sinucleína/química , Humanos , Fosfatidilcolinas/química , Fosfatidilserinas/químicaRESUMO
Pathological tau correlates well with cognitive impairments in Alzheimer's disease (AD) patients and therefore represents a promising target for immunotherapy. Targeting an appropriate B cell epitope in pathological tau could in theory produce an effective reduction of pathology without disrupting the function of normal native tau. Recent data demonstrate that the N-terminal region of tau (aa 2-18), termed the "phosphatase activation domain (PAD)", is hidden within native Tau in a 'paperclip'-like conformation. Conversely, PAD is exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and tau polymerization. Thus, we hypothesized that anti-tau2-18 antibodies may safely and specifically reduce pathological tau and prevent further aggregation, which in turn would neutralize tau toxicity. Therefore, we evaluated the immunogenicity and therapeutic efficacy of our MultiTEP platform-based vaccine targeting tau2-18 formulated with AdvaxCpG adjuvant (AV-1980R/A) in PS19 tau transgenic mice. The AV-1980R/A induced extremely high antibody responses and the resulting sera recognized neurofibrillary tangles and plaque-associated dystrophic neurites in AD brain sections. In addition, under non-denaturing conditions AV-1980R/A sera preferentially recognized AD-associated tau. Importantly, vaccination also prevented age-related motor and cognitive deficits in PS19 mice and significantly reduced insoluble total and phosphorylated tau species. Taken together, these findings suggest that predominantly targeting misfolded tau with AV-1980R/A could represent an effective strategy for AD immunotherapy.
Assuntos
Epitopos/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Vacinas/imunologia , Proteínas tau/imunologia , Animais , Formação de Anticorpos , Imunoterapia , Camundongos , Emaranhados Neurofibrilares/imunologia , Fosforilação , Proteínas tau/químicaRESUMO
BACKGROUND: Alzheimer disease (AD) is characterized by the accumulation of beta-amyloid (Aß) plaques and neurofibrillary tangles composed of hyperphosphorylated tau, which together lead to neurodegeneration and cognitive decline. Current therapeutic approaches have primarily aimed to reduce pathological aggregates of either Aß or tau, yet phase 3 clinical trials of these approaches have thus far failed to delay disease progression in humans. Strong preclinical evidence indicates that these two abnormally aggregated proteins interact synergistically to drive downstream neurodegeneration. Therefore, combinatorial therapies that concurrently target both Aß and tau might be needed for effective disease modification. METHODS: A combinatorial vaccination approach was designed to concurrently target both Aß and tau pathologies. Tau22/5xFAD (T5x) bigenic mice that develop both pathological Aß and tau aggregates were injected intramuscularly with a mixture of two MultiTEP epitope vaccines: AV-1959R and AV-1980R, targeting Aß and tau, respectively, and formulated in AdvaxCpG, a potent polysaccharide adjuvant. Antibody responses of vaccinated animals were measured by ELISA, and neuropathological changes were determined in brain homogenates of vaccinated and control mice using ELISA and Meso Scale Discovery (MSD) multiplex assays. RESULTS: T5x mice immunized with a mixture of Aß- and tau-targeting vaccines generated high Aß- and tau-specific antibody titers that recognized senile plaques and neurofibrillary tangles/neuropil threads in human AD brain sections. Production of these antibodies in turn led to significant reductions in the levels of soluble and insoluble total tau, and hyperphosphorylated tau as well as insoluble Aß42, within the brains of bigenic T5x mice. CONCLUSIONS: AV-1959R and AV-1980R formulated with AdvaxCpG adjuvant are immunogenic and therapeutically potent vaccines that in combination can effectively reduce both of the hallmark pathologies of AD in bigenic mice. Taken together, these findings warrant further development of this vaccine technology for ultimate testing in human AD.
Assuntos
Doença de Alzheimer/terapia , Vacinas contra Alzheimer , Peptídeos beta-Amiloides/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologiaRESUMO
Nanoimaging methods, atomic force microscopy (AFM) in particular, are widely used to study the interaction of biological molecules with the supported lipid bilayer (SLB), which itself is a traditional model for cellular membranes. Success in these studies is based on the availability of a stable SLB for the required observation period, which can extend several hours. The application of AFM requires that the SLB have a smooth morphology, thus enabling visualization of proteins and other molecules on its surface. Herein, we describe protocols for SLB assembly by using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) on a mica support. Our methodology enables us to assemble defect-free POPC and POPS SLBs that remain stable for at least 8 h. The application of such smooth and stable surfaces is illustrated by monitoring of the on-surface aggregation of amyloid proteins with the use of time-lapse AFM.
Assuntos
Bicamadas Lipídicas/química , Microscopia de Força Atômica/métodos , Silicatos de Alumínio/química , Fosfatidilcolinas/química , Reprodutibilidade dos Testes , Imagem com Lapso de Tempo , alfa-Sinucleína/químicaRESUMO
APOBEC3G (A3G) belongs to the family of cytosine deaminases that play an important role in the innate immune response. Similar to other, two-domain members of the APOBEC family, A3G is prone to concentration-dependent oligomerization, which is an integral for its function in the cell. It is shown that oligomerization of A3G is related to the packing mechanism into virus particle and, is critical for the so-called roadblock model during reverse transcription of proviral ssDNA. The role of oligomerization for deaminase activity of A3G is widely discussed in the literature; however, its relevance to deaminase activity for different oligomeric forms of A3G remains unclear. Here, using Atomic Force Microscopy, we directly visualized A3G-ssDNA complexes, determined their yield and stoichiometry and in parallel, using PCR assay, measured the deaminase activity of these complexes. Our data demonstrate a direct correlation between the total yield of A3G-ssDNA complexes and their total deaminase activity. Using these data, we calculated the relative deaminase activity for each individual oligomeric state of A3G in the complex. Our results show not only similar deaminase activity for monomer, dimer and tetramer of A3G in the complex, but indicate that larger oligomers of A3G retain their deaminase activity.
Assuntos
Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/metabolismo , Multimerização Proteica , Desaminase APOBEC-3G/genética , Ativação Enzimática , Humanos , Microscopia de Força Atômica/métodos , Ligação ProteicaRESUMO
BACKGROUND: The experience from clinical trials indicates that anti-Aß immunotherapy could be effective in early/pre-clinical stages of AD, whereas at the late stages promoting the clearing of Aß alone may be insufficient to halt the disease progression. At the same time, pathological tau correlates much better with the degree of dementia than Aß deposition. Therefore, targeting pathological tau may provide a more promising approach for the treatment of advanced stages of AD. Recent data demonstrates that the N-terminal region of tau spanning aa 2-18 termed "phosphatase activation domain" that is normally hidden in the native protein in 'paperclip'-like conformation, becomes exposed in pathological tau and plays an essential role in the inhibition of fast axonal transport and in aggregation of tau. Hence, we hypothesized that anti-Tau2-18 monoclonal antibodies (mAb) may recognize pathological, but not normal tau at very early stages of tauopathy and prevent or decrease the aggregation of this molecule. METHODS: Mouse mAbs were generated using standard hybridoma methodology. CDR grafting was used for humanization of mouse mAb. Humanized mAb (Armanezumab) was characterized and tested in vitro/ex vivo/in vivo using biochemical and immunological methods (HPLC, Biacore, ELISA, IHC, FRET, etc.). Stable DG44 cell line expressing Armanezumab was generated by clone selection with increased concentrations of methotrexate (MTX). RESULTS: A panel of mouse mAbs was generated, clone 1C9 was selected based on binding to pathological human tau with high affinity and humanized. Fine epitope mapping revealed conservation of the epitope of human tau recognized by the parent murine mAb and Armanezumab. Importantly, Armanezumab (i) bound to tau with high affinity as determined by Biacore; (ii) bound pathological tau in brains from AD, FTD and Pick's disease cases; (iii) inhibited seeding effect of aggregated tau from brain lysate of P301S Tg mice; (iv) inhibited cytotoxic effect of tau oligomers; (v) reduced total tau (HT7) and AT100, PHF1, AT8, AT180, p212, p214-positive tau species in brains of tau transgenic mice after intracranial injection. A stable CHO cell line producing >1.5 g/l humanized mAb, Armanezumab was generated. CONCLUSION: These findings suggest that Armanezumab could be therapeutic in clinical studies for treatment of AD.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Encéfalo/efeitos dos fármacos , Tauopatias , Proteínas tau/antagonistas & inibidores , Animais , Especificidade de Anticorpos , Humanos , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: By the time clinical symptoms of Alzheimer's disease (AD) manifest in patients there is already substantial tau pathology in the brain. Recent evidence also suggests that tau pathology can become self-propagating, further accelerating disease progression. Over the last decade several groups have tested the efficacy of protein-based anti-tau immunotherapeutics in various animal models of tauopathy. Here we report on the immunological and therapeutic potency of the first anti-tau DNA vaccine based on the MultiTEP platform, AV-1980D, in THY-Tau22 transgenic mice. METHODS: Starting at 3months of age, mice were immunized intramuscularly with AV-1980D vaccine targeting a tau B cell epitope spanning aa2-18 followed by electroporation (EP). Humoral and cellular immune responses in vaccinated animals were analyzed by ELISA and ELISpot, respectively. Neuropathological changes in the brains of experimental and control mice were then analyzed by biochemical (WB and ELISA) and immunohistochemical (IHC) methods at 9months of age. RESULTS: EP-mediated AV-1980D vaccinations of THY-Tau22 mice induced activation of Th cells specific to the MultiTEP vaccine platform and triggered robust humoral immunity response specific to tau. Importantly, no activation of potentially harmful autoreactive Th cell responses specific to endogenous tau species was detected. The maximum titers of anti-tau antibodies were reached after two immunizations and remained slightly lower, but steady during five subsequent monthly immunizations. Vaccinations with AV-1980D followed by EP significantly reduced total tau and pS199 and AT180 phosphorylated tau levels in the brains extracts of vaccinated mice, but produced on subtle non-significant effects on other phosphorylated tau species. CONCLUSIONS: These data demonstrate that MultiTEP-based DNA epitope vaccination targeting the N-terminus of tau is highly immunogenic and therapeutically potent in the THY-Tau22 mouse model of tauopathy and indicate that EP-mediated DNA immunization is an attractive alternative to protein-based adjuvanted vaccines for inducing high concentrations of anti-tau antibodies.