Elin Knudsen

An Attenuated Immune Response Is Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse Model Immunized with Amyloid- Derivatives

Immunization with amyloid-β (Aβ) 1-42 has been shown to reduce amyloid burden and improve cognition in Alzheimers disease (AD) model mice. In a human trial, possible cognitive benefit was found but in association with significant toxicity in a minority of patients. We proposed that immunization with nonfibrillogenic Aβ derivatives is much less likely to produce toxicity and have previously shown that one such derivative (K6Aβ1-30) can reduce amyloid burden in mice to a similar extent as Aβ1-42. Here, we immunized AD model mice (Tg2576) with Aβ1-30[E18E19] or with K6Aβ1-30[E18E19]. These peptides were designed to be nontoxic and to produce less T-cell response, which has been linked to toxicity. K6Aβ1-30[E18E19] induced primarily an IgM response, whereas Aβ1-30[E18E19] induced an IgG titer that was lower than previously seen with K6Aβ1-30 or Aβ1-42. However, both treated animal groups performed better than Tg controls in the radial arm maze. Amyloid burden was similar in Aβ1-30[E18E19]-vaccinated mice and their Tg controls, whereas the number of medium and small sized plaques was reduced (29-34%) in K6Aβ1-30[E18E19]-immunized mice compared with Tg controls. Amyloid burden in these mice correlated inversely with plasma IgM levels. The cognitive benefit and amyloid reduction in the K6Aβ1-30[E18E19]-vaccinated mice are likely to be related to peripheral clearance of Aβ, because IgM does not cross the blood-brain barrier because of its large size. Our results indicate that these nontoxic Aβ derivatives produce an attenuated antibody response, which is less likely to be associated with negative side effects while having cognitive benefits.

Mucosal vaccination delays or prevents prion infection via an oral route

In recent years major outbreaks of prion disease linked to oral exposure of the prion agent have occurred in animal and human populations. These disorders are associated with a conformational change of a normal protein, PrP(C) (prion protein cellular), to a toxic and infectious form, PrP(Sc) (prion protein scrapie). None of the prionoses currently have an effective treatment. A limited number of active immunization approaches have been shown to slightly prolong the incubation period of prion infection. Active immunization in wild-type animals is hampered by auto-tolerance to PrP and potential toxicity. Here we report that mucosal vaccination with an attenuated Salmonella vaccine strain expressing the mouse PrP, is effective at overcoming tolerance to PrP and leads to a significant delay or prevention of prion disease in mice later exposed orally to the 139A scrapie strain. This mucosal vaccine induced gut anti-PrP immunoglobulin (Ig)A and systemic anti-PrP IgG. No toxicity was evident with this vaccination approach. This promising finding suggests that mucosal vaccination may be a useful method for overcoming tolerance to PrP and preventing prion infection among animal and potentially human populations at risk.

Vaccination of Alzheimer’s model mice with Aβ derivative in alum adjuvant reduces Aβ burden without microhemorrhages

Immunotherapy holds great promise for Alzheimers disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. The meningoencephalitis observed in the first AD vaccination trial was likely related to excessive cell‐mediated immunity caused by the immunogen, amyloid‐β (Aβ) 1–42, and the adjuvant, QS−21. To avoid this toxicity, we have been using Aβ derivatives in alum adjuvant that promotes humoral immunity. Other potential side effects of immunotherapy are increased vascular amyloid and associated microhemorrhages that may be related to rapid clearance of parenchymal amyloid. Here, we determined if our immunization strategy was associated with this form of toxicity, and if the therapeutic effect was age‐dependent. Tg2576 mice and wild‐type littermates were immunized from 11 or 19 months and their behaviour evaluated prior to killing at 24 months. Subsequently, plaque‐ and vascular‐Aβ burden, Aβ levels and associated pathology was assessed. The therapy started at the cusp of amyloidosis reduced cortical Aβ deposit burden by 31% and Aβ levels by 30–37%, which was associated with cognitive improvements. In contrast, treatment from 19 months, when pathology is well established, was not immunogenic and therefore did not reduce Aβ burden or improve cognition. Significantly, the immunotherapy in the 11–24 months treatment group, that reduced Aβ burden, did not increase cerebral bleeding or vascular Aβ deposits in contrast to several Aβ antibody studies. These findings indicate that our approach age‐dependently improves cognition and reduces Aβ burden when used with an adjuvant suitable for humans, without increasing vascular Aβ deposits or microhemorrhages.

A non-toxic ligand for voxel-based MRI analysis of plaques in AD transgenic mice

Amyloid plaques are a characteristic feature in Alzheimers disease (AD). A novel non-toxic contrast agent is presented, Gd-DTPA-K6Abeta1-30, which is homologous to Abeta, and allows plaque detection in vivo. microMRI was performed on AD model mice and controls prior to and following intracarotid injection with Gd-DTPA-K6Abeta1-30 in mannitol solution, to transiently open the blood-brain barrier. A gradient echo T2(*)-weighted sequence was used to provide 100 microm isotropic resolution with imaging times of 115 min. The scans were examined with voxel-based analysis (VBA) using statistical parametric mapping, for un-biased quantitative comparison of ligand-injected mice and controls. The results indicate that: (1) Gd-DTPA-K6Abeta1-30 is an effective, non-toxic, ligand for plaque detection when combined with VBA (p< or =0.01-0.001), comparing pre and post-ligand injection scans. (2) Large plaques can be detected without the use of a contrast agent and this detection co-localizes with iron deposition. (3) Smaller, earlier plaques require contrast ligand for MRI visualization. Our ligand when combined with VBA may be useful for following therapeutic approaches targeting amyloid in transgenic mouse models.

Diminished amyloid-beta burden in Tg2576 mice following a prophylactic oral immunization with a salmonella-based amyloid-beta derivative vaccine.

Immunotherapy holds great promise for Alzheimers disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. Prior to the side effects in the first Elan/Wyeth AD vaccine trial, we proposed using amyloid-beta (Abeta) derivatives as a safer approach. The route of administration may also affect vaccine safety. To assess the feasibility of oral immunization that promotes mucosal immunity, Tg2576 AD model mice were treated prophylactically three times over 6 weeks starting at 3-5 months of age with a Salmonella vaccine expressing K6Abeta(1-30). At 22-24 months of age, cortical Abeta plaque burden and total Abeta(40/42) levels were reduced by 48-75% in the immunized mice compared to controls, which received unmodified Salmonella. Plaque clearance was not associated with increased microglial activation, which may be explained by the long treatment period. Furthermore, cerebral microhemorrhages were not increased in the treated mice in contrast to several passive Abeta antibody studies. These results further support our findings with this immunogen delivered subcutaneously and demonstrate its efficacy when given orally, which may provide added benefits for human use.

Diminished Amyloid-β Burden in Tg2576 Mice Following a Prophylactic Oral Immunization with a Salmonella-Based Amyloid-β Derivative Vaccine

Immunotherapy holds great promise for Alzheimers disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. Prior to the side effects in the first Elan/Wyeth AD vaccine trial, we proposed using amyloid-beta (Abeta) derivatives as a safer approach. The route of administration may also affect vaccine safety. To assess the feasibility of oral immunization that promotes mucosal immunity, Tg2576 AD model mice were treated prophylactically three times over 6 weeks starting at 3-5 months of age with a Salmonella vaccine expressing K6Abeta(1-30). At 22-24 months of age, cortical Abeta plaque burden and total Abeta(40/42) levels were reduced by 48-75% in the immunized mice compared to controls, which received unmodified Salmonella. Plaque clearance was not associated with increased microglial activation, which may be explained by the long treatment period. Furthermore, cerebral microhemorrhages were not increased in the treated mice in contrast to several passive Abeta antibody studies. These results further support our findings with this immunogen delivered subcutaneously and demonstrate its efficacy when given orally, which may provide added benefits for human use.

P1-22 Effet de la vaccination par un dérivé du peptide Aβ, K6Aβ1-30, chez des microcèbes âgés

L’immunotherapie contre la maladie d’Alzheimer (MA) est une approche therapeutique tres prometteuse. En effet, chez les souris transgeniques, l’immunisation par le peptide amyloide β (Aβ1-42) induit une reponse immunitaire, ameliore la cognition et reduit la charge amyloide cerebrale. Cependant, une encephalopathie peut survenir chez certains patients. C’est pourquoi des derives de ce peptide sont actuellement testes comme nouveaux immunogenes afin de diminuer les risques inflammatoires. Objectifs notre but est d’etudier les effets du peptide modifie K6Aβ1-30 sur des microcebes âges qui peuvent developper des lesions de type Alzheimer. Ces animaux ont ete suivis via des controles sanguins, des etudes comportementales et des examens histologiques post mortem. Materiel et methodes trente-deux microcebes âges de 6-10 ans (temoins versus immunises) ont ete evalues par une tâche a choix multiples mettant en jeu la memoire spatiale : memorisation d’un trajet dans un labyrinthe. Le groupe immunise a ensuite recu 3 injections sous-cutanees de K6Aβ1-30 suivies de prelevements sanguins a differents temps post-injection afin de suivre la reponse immunitaire. Le comportement des animaux a ete teste en parallele. A la fin du protocole, des analyses neurohistologiques ont ete effectuees pour rechercher les lesions de type Alzheimer : plaques amyloides, proteine Tau agregee, gliose… Resultats Les microcebes âges presentent une bonne reponse immunitaire. De plus, 5 mois apres la derniere injection, la reponse immunitaire etait reversible en l’absence d’immunisation. L’analyse comportementale montre un maintien des competences mnesiques chez les animaux immunises. L’analyse neuropathologique montre une diminution des lesions corticales. Des plaques amyloides compactes ont ete detectees chez un animal temoin alors que chez un animal immunise les plaques etaient diffuses. Nous avons egalement observe une diminution de l’Aβ intracellulaire chez les microcebes immunises accompagnee d’une augmentation du nombre de neurones pyramidaux marques par la proteine tau normale. D’autre part, aucune microhemorragie n’a ete detectee chez les animaux et la presence de microglie est plus faible chez les immunises. Conclusion la vaccination par le peptide K6As1-30, bien toleree par les microcebes âges, va dans le sens d’une amelioration des performances cognitives et d’une diminution de la presence de marqueurs pathologiques.

Diminished Aβ Burden in Tg2576 Mice Following a Prophylactic Oral Immunization with a Salmonella-based Aβ Derivative Vaccine

Immunotherapy holds great promise for Alzheimers disease (AD) and other conformational disorders but certain adverse reactions need to be overcome. Prior to the side effects in the first Elan/Wyeth AD vaccine trial, we proposed using amyloid-beta (Abeta) derivatives as a safer approach. The route of administration may also affect vaccine safety. To assess the feasibility of oral immunization that promotes mucosal immunity, Tg2576 AD model mice were treated prophylactically three times over 6 weeks starting at 3-5 months of age with a Salmonella vaccine expressing K6Abeta(1-30). At 22-24 months of age, cortical Abeta plaque burden and total Abeta(40/42) levels were reduced by 48-75% in the immunized mice compared to controls, which received unmodified Salmonella. Plaque clearance was not associated with increased microglial activation, which may be explained by the long treatment period. Furthermore, cerebral microhemorrhages were not increased in the treated mice in contrast to several passive Abeta antibody studies. These results further support our findings with this immunogen delivered subcutaneously and demonstrate its efficacy when given orally, which may provide added benefits for human use.

AB derivatives as potentially safer vaccines for Alzheimer’s disease

based on an extensive preclinical literature which shows that AL-108 has potent neuroprotective, memory enhancing and neurotrophic properties. Furthermore, the data from these studies provide insights into a proposed mechanism of action of AL-108 against two primary pathologies of AD beta amyloid toxicity and microtubule breakdown that is associated with tau hyperphosphorylation. Objectives: To evaluate AL-108 in preclinical and toxicological studies toward clinical trials in Alzheimer’s disease patients. Methods and Results: Neuroprotective activities of AL-108 have been investigated in vitro against: The AD toxin ( amyloid peptide); The toxic envelope protein of the human immunodeficiency virus (HIV; gp120); Glucose deprivation; Electrical blockade (tetrodotoxin); Oxidative stress (hydrogen peroxide and glutathione); Dopamine toxicity and excitotoxicity (N-methyl-D-aspartate). AL-108 has also been evaluated as a potential neuroprotectant in a variety of animal models related to AD: The rat cholinotoxicity model; Apolipoprotein E-deficient mice; Mouse head trauma model; Hypertensive rat stroke model. AL-108 is an eight amino acid peptide with the following characteristics: It has a lipophilic structure that allows penetration through lipid membranes as in the case of the cellular membrane and the blood brain barrier; It has intrinsic sheet breaker characteristics, thus acting as a peptide chaperone to protect against toxic A plaque associated with AD; It chelates trace amounts of heavy metals, thus preventing toxicity; It binds to tubulin and promotes proper microtubule assembly as a peptide chaperone. In conclusion, based on the preclinical results toxicology studies have been peroformed. Good Laboratory Practice (GLP) animal studies have demonstrated that AL-108 is safely tolerated, brain bioavailable by intranasal, intravenous and subcutaneous routes and suitable for drug development. Allon Therapeutics has filed an Investigational New Drug application with the United States Food and Drug Administration for AL-108 and has received FDA approval to proceed.

In vivo detection of amyloid plaques in AD transgenic mice using gadolinium labeled ligands by MRI

administration. Binding specificity to amyloid plaque was evaluated with tissue homogenates from the postmortem brain (frontal cortex and cerebellum) from Alzheimer’s patients and control subjects. The tissue homogenates were incubated in PBS and 0.1% fetal bovine serum with radiotracers for 15 min, centrifuged and bound radioactivity measured. Brain tissue sections were incubated with radiotracers, washed and exposed to Storage Phosphor Screens. Adjacent sections stained with Congo red. Results: The octanol-water partition coefficient (log PC) was in the range (1-3) optimum for blood brain barrier transport. The uptake of the compounds in the normal mouse brain was rapid and so was the clearance. Mouse brain uptake (%ID/g SD) at 2 min was 3.14 0.71 and 1.39 0.29 for CQ and HQ respectively and in 1 h decreased by 98%. Blood activity was 9.5 2.12 and 4.73 1.09 at 2 min for CQ and HQ respectively and decreased by 96% at 1 h. Practically no radioactivity was detectable in the brain and blood at 24 h for both the tracers. Activity in the frontal cortex of the human AD brain was 400-500% of the cerebellum and 300-400% of control cortex. Autoradiography showed the localization of the tracers in regions containing amyloid plaques recognized by Congo red. Nanoparticles enhanced the brain uptake by 15% compared to the free drug. Conclusions: Radioiodinated quinoline derivatives have rapid brain uptake and fast clearance from the normal brain and blood in mice. They showed binding specificity to amyloid deposits in AD brain homogenates. Plaque deposits were visualized by autoradiography. These studies warrant further evaluation of this class of molecules as amyloid imaging agents.