Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy.

BackgroundMultiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation.ResultsIn this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells.ConclusionsThis study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies

Tailoring the Antibody Response to Aggregated Aß Using Novel Alzheimer-Vaccines

Recent evidence suggests Alzheimer-Disease (AD) to be driven by aggregated Aß. Capitalizing on the mechanism of molecular mimicry and applying several selection layers, we screened peptide libraries for moieties inducing antibodies selectively reacting with Aß-aggregates. The technology identified a pool of peptide candidates; two, AFFITOPES AD01 and AD02, were assessed as vaccination antigens and compared to Aβ1-6, the targeted epitope. When conjugated to Keyhole Limpet Hemocyanin (KLH) and adjuvanted with aluminum, all three peptides induced Aß-targeting antibodies (Abs). In contrast to Aß1-6, AD01- or AD02-induced Abs were characterized by selectivity for aggregated forms of Aß and absence of reactivity with related molecules such as Amyloid Precursor Protein (APP)/ secreted APP-alpha (sAPPa). Administration of AFFITOPE-vaccines to APP-transgenic mice was found to reduce their cerebral amyloid burden, the associated neuropathological alterations and to improve their cognitive functions. Thus, the AFFITOME-technology delivers vaccines capable of inducing a distinct Ab response. Their features may be beneficial to AD-patients, a hypothesis currently tested within a phase-II-study

Distribution of Alarin Immunoreactivity in the Mouse Brain

Alarin is a 25 amino acid peptide that belongs to the galanin peptide family. It is derived from the galanin-like peptide gene by a splice variant, which excludes exon 3. Alarin was first identified in gangliocytes of neuroblastic tumors and later shown to have a vasoactive function in the skin. Recently, alarin was demonstrated to stimulate food intake as well as the hypothalamic–pituitary–gonadal axis in rodents, suggesting that it might be a neuromodulatory peptide in the brain. However, the individual neurons in the central nervous system that express alarin have not been identified. Here, we determined the distribution of alarin-like immunoreactivity (alarin-LI) in the adult murine brain. The specificity of the antibody against alarin was demonstrated by the absence of labeling after pre-absorption of the antiserum with synthetic alarin peptide and in transgenic mouse brains lacking neurons expressing the GALP gene. Alarin-LI was observed in different areas of the murine brain. A high intensity of alarin-LI was detected in the accessory olfactory bulb, the medial preoptic area, the amygdala, different nuclei of the hypothalamus such as the arcuate nucleus and the ventromedial hypothalamic nucleus, the trigeminal complex, the locus coeruleus, the ventral chochlear nucleus, the facial nucleus, and the epithelial layer of the plexus choroideus. The distinct expression pattern of alarin in the adult mouse brain suggests potential functions in reproduction and metabolism

Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy.

BackgroundMultiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation.ResultsIn this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells.ConclusionsThis study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies

Journal of Spatial Science / Evaluation of modified Interferon alpha mRNA constructs for the treatment of non-melanoma skin cancer

Application of in vitro transcribed (IVT) messenger ribonucleic acid (mRNA) is an increasingly popular strategy to transiently produce proteins as therapeutics in a tissue or organ of choice. Here, we focused on the skin and aimed to test if whole human skin tissue explant technology can be used to evaluate the expression efficacy of different IVT Interferon alpha (IFN-) mRNA constructs in situ, after biolistic delivery. Skin explants were viable and intact for at least five days based on histologic analysis and TUNEL staining. Using GFP reporter mRNA formulations, we found mostly epidermal expression after biolistic delivery. Two out of five sequence-optimized IFN- mRNA variants resulted in significantly improved IFN- protein expression in human skin compared to native IFN- mRNA transfection. IFN- secretion analysis of the surrounding culture media confirmed these results. We provide a proof-of-concept that IFN- mRNA delivery into intact human full thickness skin explants can be utilized to test mRNA sequence modifications ex vivo. This approach could be used to develop novel mRNA-based treatments of common epidermal skin conditions including non-melanoma skin cancer, where IFN- protein therapy has previously shown a strong therapeutic effect.(VLID)286371

Effects of single and combined immunotherapy approach targeting amyloid β protein and α‐synuclein in a dementia with Lewy bodies–like model

IntroductionImmunotherapeutic approaches targeting amyloid β (Aβ) protein and tau in Alzheimer's disease and α-synuclein (α-syn) in Parkinson's disease are being developed for treating dementia with Lewy bodies. However, it is unknown if single or combined immunotherapies targeting Aβ and/or α-syn may be effective.MethodsAmyloid precursor protein/α-syn tg mice were immunized with AFFITOPEs® (AFF) peptides specific to Aβ (AD02) or α-syn (PD-AFF1) and the combination.ResultsAD02 more effectively reduced Aβ and pTau burden; however, the combination exhibited some additive effects. Both AD02 and PD-AFF1 effectively reduced α-syn, ameliorated degeneration of pyramidal neurons, and reduced neuroinflammation. PD-AFF1 more effectively ameliorated cholinergic and dopaminergic fiber loss; the combined immunization displayed additive effects. AD02 more effectively improved buried pellet test behavior, whereas PD-AFF1 more effectively improved horizontal beam test; the combined immunization displayed additive effects.DiscussionSpecific active immunotherapy targeting Aβ and/or α-syn may be of potential interest for the treatment of dementia with Lewy bodies

AFFITOPE immunization reduces cerebral amyloid levels in Tg2576 mice (ELISA).

<p>Groups of Tg2576 mice (n = 10/group) received 6 monthly injections of KLH/ALUM or AD01-, AD02-conjugate vaccines. Brains were isolated, 8 weeks after the 6^th immunization, extracted and soluble and insoluble brain fractions were subjected to Human Aß40 and Human Aß42 ELISA (EMD-Milipore, USA) analysis. Neither AD01- (A) nor AD02 treated animals (B) showed a significant change of soluble Aß1-40 and Aß1-42 following immunotherapy as compared to control immunized animals. Insoluble Aß was reduced significantly following immunotherapy. C) AD01 treated animals showed a 69% reduction of Aß1-40 levels (p = 0.005) and a 78% reduction of Aß1-42 (p = 0.015), respectively. D) For AD02 a 60% reduction of Aß1-40 (p = 0.033) and a 62% (p = 0.056) reduction of Aß1-42 could be detected. Results are expressed as average ± SEM and are given as ng/mg total protein. Black bars represent Aß1-40 and white bars represent Aß1-42 values. Asterisks in C+D indicate statistical significant difference (*…p<0.05, **…p<0.01);</p

AD01 and AD02 immunization does not induce self-reactive T-Cells.

<p>Neither AD01 nor AD02 treated mice showed any sign of Aß-specific T-cell activation in two ELISPOT assays (A+B). Re-stimulation using the carrier (KLH) was resulting in a stimulation of IL4 and Interferon gamma (INFg) secretion, indicative of the presence of carrier specific T-cells following immunization with AD01 and AD02. The positive control Ovalbumin was able to induce a slightly higher Interferon gamma secretion than the carrier used in the AFFITOPE vaccines (B). A+B depict two representative ELISPOT analyses following vaccination of Ovalbumin, AD01 and AD02. A) IL4 secretion following splenocyte restimulation using carrier (KLH) and Aß compared to the controls OVA244 (TEWTSSNVMEERKIKV; MHC class II restricted to demonstrate Ovalbumin induced T-cells) and PMA/ionomycin (PMA/Ion); B) IFNg secretion following splenocyte restimulation compared to the positive controls OVA245 (SIINFEKL; MHC class I restricted to demonstrate Ovalbumin induced T-cells) and Concavalin A (ConA). Bg describes the background of secretion in non-stimulated cells in this assay. Numbers are the total number of spots per million of cells seeded on the ELISPOT plates.</p