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

A Dual-Pathogen Mitral Valve Endocarditis Caused by <i>Coxiella burnetii</i> and <i>Streptococcus gordonii</i>—Which Came First?

Infective endocarditis (IE) is still a life-threatening disease with high morbidity and mortality. While usually caused by a single bacterium, poly-microbial infective endocarditis (IE) is rare. Here, we report a (blood-culture-negative) dual pathogen mitral valve IE caused by Coxiella burnetii and Streptococcus gordonii: A 53-year-old woman was presented to an internal medicine department with abdominal pain for further evaluation. Within the diagnostic work up, transthoracic echocardiography (TTE) revealed an irregularly shaped echogenic mass (5 × 13 mm) adherent to the edge of the posterior mitral valve leaflet and protruding into the left atrium. As infected endocarditis was suspected, blood cultures were initially obtained, but they remained negative. Chronic Q fever infection was diagnosed using serologic testing. After the occurrence of cerebral thromboembolic events, the patient was admitted for mitral valve surgery. Intraoperatively, a massively destructed mitral valve with adhering vegetations was noted. Examination of the mitral valve by broad-range bacterial polymerase chain reaction (PCR) and amplicon sequencing confirmed Coxiella burnetii infection and yielded Streptococcus gordonii as the second pathogen. Based on the detailed diagnosis, appropriate antibiotic therapy of both pathogens was initiated, and the patient could be discharged uneventfully on the 11th postoperative day after a successful minimal-invasive mitral valve replacement

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

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

Analysis of the immune response following injection of AD01, AD02 and Aß1-6 conjugate vaccines.

<p>Mice were s.c. injected 6 times at a 4-week interval with AD01 (n = 9), AD02 (n = 8) and Aß1-6 conjugate (n = 9) adsorbed to aluminum hydroxide (ALUM). Plasma was taken in monthly intervals and at sacrification. Samples were analyzed for their concentration of IgG Abs against specific peptides. Values depicted are the titer calculated as OD max/2 (at 405nm) plus SEM unless otherwise stated. A) IgG response torwards the respective immunizing peptide (AD01: anti AD01; AD02: anti AD02, Aβ1-6: anti Aβ1-6); B) Reactivity towards human Aβ1-10 after immunization with AD01-, AD02- and Aβ1-6-based conjugate vaccines. Note, none of the 3 vaccines elicits Abs that would react with the Aβ11-19, used as a specificity control (not shown); C) Kinetics of the IgG responses to the immunizing peptide following vaccination with AD01-, AD02- or Aβ1-6 conjugates (AD01… black circle, AD02… black quadrat, Aß1-6… black triangle); D) Ratio of AD02-induced peptide-specific IgG in CSF and plasma. Analysis of AD02-specific IgG levels in CSF and plasma in 13 AD02-immunized animals revealed an average ratio of 0.31% (+/- 0.05%). E) Analysis of sera from vaccinated animals regarding their reactivity towards murine Aβ1-42. Only Aβ1-6 immunized animals show a relevant cross-reactivity to murine Aβ1-42 (Aβ1-6 (n = 9) vs. AD01 (n = 10); p<0.05 and Aβ1-6 (n = 9) vs. AD02 (n = 28); p<0.01); F) IgG response towards the respective immunizing peptide (AD01: anti AD01; AD02: anti AD02) compared to the respective other AFFITOPE peptide (AD01: anti AD02; AD02: anti AD01). Animals included: n = 9 for AD01, n = 8 for AD02.</p