Efficient Expression of an Alzheimer's Disease Vaccine Candidate in the Microalga Schizochytrium sp. Using the Algevir System.

Alzheimer's disease (AD) is the most common neurodegenerative disease, where ß-amyloid (Aß) plays a key role in forming conglomerated senile plaques. The receptor of advanced glycation end products (RAGE) is considered a therapeutic target since it transports Aß into the central nervous system, favoring the pathology progression. Due to the lack of effective therapies for AD, several therapeutic approaches are under development, being vaccines considered a promising alternative. Herein, the use of the Algevir system was explored to produce in the Schizochytrium sp. microalga the LTB:RAGE vaccine candidate. Algevir relies in an inducible geminiviral vector and led to yields of up to 380 µg LTB:RAGE/g fresh weight biomass at 48-h post-induction. The Schizochytrium-produced LTB:RAGE vaccine retained its antigenic activity and was highly stable up to temperatures of 60 °C. These data demonstrate the potential of Schizochytrium sp. as a platform for high production of thermostable recombinant antigens useful for vaccination against AD.

Effect of amyloid-ß (Aß) immunization on hyperphosphorylated tau: a potential role for glycogen synthase kinase (GSK)-3ß.

AIMS: Active amyloid-ß (Aß) immunotherapy in Alzheimer's disease (AD) induces removal of Aß and phosphorylated tau (ptau). Glycogen synthase kinase (GSK)-3ß is a kinase, responsible for phosphorylation of tau, activation of which can be induced by phosphorylated double-stranded RNA-dependent protein kinase (pPKR). Using a post-mortem cohort of immunized AD cases, we investigated the effect of Aß immunization on GSK-3ß expression and pPKR. METHODS: We immunostained 11 immunized AD cases and 28 unimmunized AD cases for active, inactive and total GSK-3ß, and for pPKR. Quantification of protein load was performed in the hippocampal region including CA1, subiculum and entorhinal cortex. RESULTS: All three areas showed a significant decrease in the three forms of GSK-3ß (P < 0.05) and a nonsignificant trend towards lower pPKR load in the immunized AD cases compared with the unimmunized AD cases. CONCLUSION: The lower GSK-3ß expression generated by Aß immunotherapy shows evidence of a modification of the signalling pathway induced by GSK-3ß leading to the overall reduction of tau, supporting the contention that in humans, GSK-3ß unifies Aß and tau-related neuropathology.

Development of a new DNA vaccine for Alzheimer disease targeting a wide range of aß species and amyloidogenic peptides.

It has recently been determined that not only Aß oligomers, but also other Aß species and amyloidogenic peptides are neurotoxic in Alzheimer disease (AD) and play a pivotal role in AD pathogenesis. In the present study, we attempted to develop new DNA vaccines targeting a wide range of Aß species. For this purpose, we first performed in vitro assays with newly developed vaccines to evaluate Aß production and Aß secretion abilities and then chose an IgL-Aßx4-Fc-IL-4 vaccine (designated YM3711) for further studies. YM3711 was vaccinated to mice, rabbits and monkeys to evaluate anti-Aß species antibody-producing ability and Aß reduction effects. It was found that YM3711 vaccination induced significantly higher levels of antibodies not only to Aß1-42 but also to AD-related molecules including AßpE3-42, Aß oligomers and Aß fibrils. Importantly, YM3711 significantly reduced these Aß species in the brain of model mice. Binding and competition assays using translated YM3711 protein products clearly demonstrated that a large part of antibodies induced by YM3711 vaccination are directed at conformational epitopes of the Aß complex and oligomers. Taken together, we demonstrate that YM3711 is a powerful DNA vaccine targeting a wide range of AD-related molecules and is worth examining in preclinical and clinical trials.

Enhancing Th2 immune responses against amyloid protein by a DNA prime-adenovirus boost regimen for Alzheimer's disease.

Accumulation of aggregated amyloid beta-protein (Abeta) in the brain is thought to be the initiating event leading to neurodegeneration and dementia in Alzheimer's disease (AD). Therefore, therapeutic strategies that clear accumulated Abeta and/or prevent Abeta production and its aggregation are predicted to be effective against AD. Immunization of AD mouse models with synthetic Abeta prevented or reduced Abeta load in the brain and ameliorated their memory and learning deficits. The clinical trials of Abeta immunization elicited immune responses in only 20% of AD patients and caused T-lymphocyte meningoencephalitis in 6% of AD patients. In attempting to develop safer vaccines, we previously demonstrated that an adenovirus vector, AdPEDI-(Abeta1-6)11, which encodes 11 tandem repeats of Abeta1-6 can induce anti-inflammatory Th2 immune responses in mice. Here, we investigated whether a DNA prime-adenovirus boost regimen could elicit a more robust Th2 response using AdPEDI-(Abeta1-6)11 and a DNA plasmid encoding the same antigen. All mice (n=7) subjected to the DNA prime-adenovirus boost regimen were positive for anti-Abeta antibody, while, out of 7 mice immunized with only AdPEDI-(Abeta1-6)11, four mice developed anti-Abeta antibody. Anti-Abeta titers were indiscernible in mice (n=7) vaccinated with only DNA plasmid. The mean anti-Abeta titer induced by the DNA prime-adenovirus boost regimen was approximately 7-fold greater than that by AdPEDI-(Abeta1-6)11 alone. Furthermore, anti-Abeta antibodies induced by the DNA prime-adenovirus boost regimen were predominantly of the IgG1 isotype. These results indicate that the DNA prime-adenovirus boost regimen can enhance Th2-biased responses with AdPEDI-(Abeta1-6)11 in mice and suggest that heterologous prime-boost strategies may make AD immunotherapy more effective in reducing accumulated Abeta.