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

Collection, Standardization and Attribution of Robust Disaster Event Information – A Demonstrator of a National Event-Based Loss and Damage Database in Austria

Loss and damage databases are essential tools within the disaster risk management cycle for making informed decisions. However, even in data-rich countries such as Austria, no consistent and curated multi-hazard database is available. Based on the requirements of the United Nations, the European Union, as well as on national demands to deal with disaster impacts, we conceived and set up a demonstrator for a consistent multi-hazard national event-based loss and damage database that addresses event identification, loss accounting and disaster forensics according to international standards. We built our database on already existing data from administration and federal agencies and formulated a process to combine those data in a synergetic way. Furthermore, we tested how earth observation and weather data could help to derive more robust disaster event information. Our demonstrator focuses on two Austrian federal provinces, three hazard types – floods, storms and mass movements – and the period between 2005 and 2018. By analyzing over 140.000 single event descriptions, we conclude that – despite some limitations in retrospective data harmonization – the implementation of a curated event-based national loss and damage database is feasible and adds significant value compared to the usage of single national datasets or existing international databases such as EM-DAT or the Risk Data Hub. With our demonstrator, we are able to support the national risk assessment, the national Sendai Monitoring and federal disaster risk management with the provision of best possible harmonized loss and damage information, tailored indicators and statistics as well as hazard impact maps on the municipality scale.ISSN:2076-326

Cerebral amyloid angiopathy and microhemorrhaging are unchanged following AFFITOPE-immunization.

<p>The analysis of the incidence of amyloid bearing vessel in AFFITOPE- and control treated animals by assessing 3A5 staining in cerebral vessels reveals no significant differences (A-C). A) Example from a cortical section of a control animal. B) Example from a cortical section of an AD02-treated animal. C) Quantitative analysis demonstrating the average number of 3A5 positive vessels/mm² (avg. +/- SEM) in control (n = 9), and AD02-treated animals (n = 8). The analysis of the incidence of cerebral micro-hemorrhages in these animals by assessing Hemosiderin staining (= Prussian Blue) did not show significant differences at 14 months of age (D-F). D) Example from a cortical section of a control animal. E) Example from a cortical section of an AD02-treated animal. F) quantitative analysis demonstrating the average number of Hemosiderin positive vessels/animal in control (n = 9), and AD02 treated animals (n = 8, respectively); Arrows indicate 3A5 positive vessels, arrowheads indicate amyloid deposits. Scale bar: 50µm; pictures taken at 20x magnification</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

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

AFFITOPE immunization reduces cerebral amyloid deposition in Tg2576 mice.

<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. Quantification of the relative total brain area covered by amyloid deposits (in % of total tissue analyzed) is based on immuno-fluorescence staining using the Aß specific mAb 3A5. Representative subregions of the cortex (A, B) and dentate gyrus (C, D) of controls (A, C) and AD02- (B, D) immunized mice are shown. E) AD02-vaccination reduces the relative area covered by amyloid deposits compared to controls by 70% (diffuse and dense cored amyloid; p˂0.05). F) AD01 vaccination reduces the relative area covered by amyloid deposits compared to controls by 62% (diffuse and dense cored amyloid; p˂0.05). Box plots in E and F show minimum, 25% percentile, median, 75% percentile and maximum. Asterisks in E+F indicate statistical significant difference (p<0.05); Arrowhead in C indicates unspecific fluorescence from a cerebral vessel. Scale bar: 200µM; pictures taken at 10x magnification</p

AFFITOPE-induced antibodies detect amyloid deposits but spare neuronal APP on murine and human brain sections.

<p>Sections prepared from the hippocampus (A, D, G, J, K, M) and the cortex (B, E, H), of a 12 month old Tg2576 mouse were incubated with plasma of AD01- (D, J) or AD02-treated mice (E, K) and, for control purposes, with the antibodies 6E10 (A+B) and mAb 22C11 (J, K, M), recognizing Aß and full length APP. G) and H) show a loss of immunoreactivity on Tg2576 brain sections incubated with AD01- and AD02-induced samples which were mixed with the respective AFFITOPE peptide to inhibit AFFITOPE specific staining (i.e. G: AD01-induced plasma + 10µM AD01 peptide, H: AD02-induced plasma + 10µM AD02 peptide). Sections prepared from the cortex of a female AD patient (C, F, I, L Braak stage VI,) were incubated with the Aß specific control antibody BAM10 (C), AD01- (F) and AD02-induced plasma (I) or plasma from naïve control mice as negative control (L). Binding of the Abs was detected using a standard DAB immuno-histochemistry protocol. The analysis of human sections reveals a specific amyloid deposit staining of AD01-(F) and AD02-induced Abs (I) present in murine plasma similar to staining obtained by using control antibody BAM10 (C), whereas no staining was detectable with plasma derived from a naïve, untreated animal (L). Pictures were taken at a magnification of 20x. Scale bars: 200µm; circles in G and H indicate amyloid deposits devoid of amyloid specific staining by AD01- or AD02- induced antibodies following peptide-specific competition.</p