Effects of single and combined immunotherapy approach targeting amyloid ß protein and α-synuclein in a dementia with Lewy bodies-like model.

INTRODUCTION: Immunotherapeutic approaches targeting amyloid ß (Aß) 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 Aß and/or α-syn may be effective. METHODS: Amyloid precursor protein/α-syn tg mice were immunized with AFFITOPEs® (AFF) peptides specific to Aß (AD02) or α-syn (PD-AFF1) and the combination. RESULTS: AD02 more effectively reduced Aß 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. DISCUSSION: Specific active immunotherapy targeting Aß and/or α-syn may be of potential interest for the treatment of dementia with Lewy bodies.

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

BACKGROUND: Multiple 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. RESULTS: In 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. CONCLUSIONS: This 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 Aß1-6, 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.

Peptide-based anti-PCSK9 vaccines - an approach for long-term LDLc management.

BACKGROUND: Low Density Lipoprotein (LDL) hypercholesterolemia, and its associated cardiovascular diseases, are some of the leading causes of death worldwide. The ability of proprotein convertase subtilisin/kexin 9 (PCSK9) to modulate circulating LDL cholesterol (LDLc) concentrations made it a very attractive target for LDLc management. To date, the most advanced approaches for PCSK9 inhibition are monoclonal antibody (mAb) therapies. Although shown to lower LDLc significantly, mAbs face functional limitations because of their relatively short in vivo half-lives necessitating frequent administration. Here, we evaluated the long-term efficacy and safety of PCSK9-specific active vaccines in different preclinical models. METHODS AND FINDING: PCSK9 peptide-based vaccines were successfully selected by our proprietary technology. To test their efficacy, wild-type (wt) mice, Ldlr+/- mice, and rats were immunized with highly immunogenic vaccine candidates. Vaccines induced generation of high-affine PCSK9-specific antibodies in all species. Group mean total cholesterol (TC) concentration was reduced by up to 30%, and LDLc up to 50% in treated animals. Moreover, the PCSK9 vaccine-induced humoral immune response persisted for up to one year in mice, and reduced cholesterol levels significantly throughout the study. Finally, the vaccines were well tolerated in all species tested. CONCLUSIONS: Peptide-based anti-PCSK9 vaccines induce the generation of antibodies that are persistent, high-affine, and functional for up to one year. They are powerful and safe tools for long-term LDLc management, and thus may represent a novel therapeutic approach for the prevention and/or treatment of LDL hypercholesterolemia-related cardiovascular diseases in humans.

Next-generation active immunization approach for synucleinopathies: implications for Parkinson's disease clinical trials.

Immunotherapeutic approaches are currently in the spotlight for their potential as disease-modifying treatments for neurodegenerative disorders. The discovery that α-synuclein (α-syn) can transmit from cell to cell in a prion-like fashion suggests that immunization might be a viable option for the treatment of synucleinopathies. This possibility has been bolstered by the development of next-generation active vaccination technology with short peptides-AFFITOPEs(®) (AFF)- that do not elicit an α-syn-specific T cell response. This approach allows for the production of long term, sustained, more specific, non-cross reacting antibodies suitable for the treatment of synucleinopathies, such as Parkinson's disease (PD). In this context, we screened a large library of peptides that mimic the C-terminus region of α-syn and discovered a novel set of AFF that identified α-syn oligomers. Next, the peptide that elicited the most specific response against α-syn (AFF 1) was selected for immunizing two different transgenic (tg) mouse models of PD and Dementia with Lewy bodies, the PDGF- and the mThy1-α-syn tg mice. Vaccination with AFF 1 resulted in high antibody titers in CSF and plasma, which crossed into the CNS and recognized α-syn aggregates. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn oligomers in axons and synapses, accompanied by reduced degeneration of TH fibers in the caudo-putamen nucleus and by improvements in motor and memory deficits in both in vivo models. Clearance of α-syn involved activation of microglia and increased anti-inflammatory cytokine expression, further supporting the efficacy of this novel active vaccination approach for synucleinopathies.

Detection of peri-synaptic amyloid-ß pyroglutamate aggregates in early stages of Alzheimer's disease and in AßPP transgenic mice using a novel monoclonal antibody.

The neurodegenerative pathology in patients with Alzheimer's disease (AD) has been associated with the progressive accumulation of aggregated and post-translationally modified amyloid-ß (Aß) species. Among them, recent studies indicate that the pyroglutamate modification of Aß (pE(3)Aß) catalyzed by glutaminyl cyclase might play an important role in the pathogenesis of AD. Although the effects of the pyroglutamate modification on Aß aggregation and toxicity have been investigated, less is known about the distribution of pE(3)Aß across the spectrum of AD and in the brains of amyloid-ß protein precursor (AßPP) transgenic (tg) animals. For this purpose, we generated a novel monoclonal antibody (denominated D129) that specifically recognizes pE(3)Aß and characterized the patterns of distribution in the postmortem brain samples from AD patients divided by disease stage (Braak stage) and in AßPP tg mice. We found that in early stages of AD and young AßPP tg mice pE(3)Aß was found in discrete linear and granular aggregates in the neuropil that co-localized with the pre-synaptic protein synaptophysin and was in close opposition to dendrites labeled with MAP2. In later stages of AD and in older AßPP tg mice, pE(3)Aß was abundant in diffuse and mature plaques. In conclusion, this study suggests that peri-synaptic accumulation of pE(3)Aß might contribute to early cognitive dysfunction in AD.

AFFITOME® technology in neurodegenerative diseases: the doubling advantage.

Neurodegenerative diseases are still an area of unmet medical need. This is in contrast to our increasing knowledge on their pathology (e.g., Alzheimer's- (AD), Parkinson's (PD) disease). They are driven by the cerebral accumulation and aggregation of specific proteins (e.g., ß-amyloid and hyperphosphorylated tau in the case of AD) in defined brain regions and, as a consequence, death of neurons. Accordingly, removal of given protein aggregates is expected to modify the course of the respective neurodegenerative disease. This has been convincingly demonstrated in animal models of human diseases. However, not every technology that can be used and proves successful in animal models can be translated to the human situation. As highlighted by recent progress in the field of AD research, specific immunotherapy is a viable option in this regard. Given the fact that the aggregates are composed of self-proteins, immunotherapeutic approaches have to consider the issue of potential autoimmunity. This is especially true in case of vaccines. An innovative solution to this problem is offered by the so called AFFITOME® technology, which relies on the use of "doubles" of native molecules, functionally mimotopes or AFFITOPES® if identified by AFFiRiS, as the antigenic vaccine component.

The artificial antimicrobial peptide KLKLLLLLKLK induces predominantly a TH2-type immune response to co-injected antigens.

Cationic antimicrobial peptides (CAMPs) are active defence components of the innate immune system. Several artificial CAMPs have been designed as antibiotic peptide therapeutics, but none have been reported to exert adjuvant activity in animal models. Here we show for the first time that an artificial CAMP, KLKLLLLLKLK (KLKL5KLK), is a potent inducer of adaptive immunity to co-injected antigens in vivo. High levels of antigen-specific antibodies were obtained after co-injection of KLKL5KLK with the model antigen ovalbumin (OVA) or a commercially available influenza vaccine. We show that KLKL5KLK induces a sustained immune response with a prevalent TH2 profile when co-injected with proteinaceous and peptide-based antigens. Furthermore, the immuno-enhancing activity of peptide KLKL5KLK was retained when C-terminally amidated or synthesised as retro-all-D-peptide. We provide evidence that KLKL5KLK enhances the association of antigen to antigen-presenting cells and forms a depot of antigen at the site of injection, making it an interesting adjuvant for novel vaccine design.

Poly-L-arginine synergizes with oligodeoxynucleotides containing CpG-motifs (CpG-ODN) for enhanced and prolonged immune responses and prevents the CpG-ODN-induced systemic release of pro-inflammatory cytokines.

This study describes an entirely synthetic vaccine composed of antigenic peptides (T cell epitopes), oligodeoxynucleotides containing CpG-motifs (CpG-ODN) and poly-L-arginine (pR). CpG-ODN are known to be potent inducers of predominantly type 1-like immune responses, while polycationic amino acids, like pR, facilitate the uptake of antigens into antigen presenting cells (APCs). We demonstrate that the application of peptides and pR/CpG-ODN results in strongly enhanced peptide-specific immune responses as compared to the application of peptides with either of the immunomodulators alone. High numbers of antigen-specific T cells can be observed even after only one injection of the vaccine for a remarkably long period of time (at least 372 days). Furthermore, the potentially harmful systemic release of pro-inflammatory cytokines induced upon injection of CpG-ODN is inhibited. Thus, the combined application of CpG-ODN and pR may represent a novel vaccine strategy in humans.

Vaccination with poly-L-arginine as immunostimulant for peptide vaccines: induction of potent and long-lasting T-cell responses against cancer antigens.

Vaccines that induce high numbers of sustained T cell responses are urgently needed for the treatment of numerous diseases including cancer. Antigen-presenting cells (APCs), the most important of which are dendritic cells, orchestrate antigen-dependent T cell responses in that they present antigens to T cells in an appropriate environment. Here we present evidence that after vaccination with a simple mixture of the cationic poly-amino acid poly-L-arginine and tumor antigen-derived peptide antigens, large numbers of antigen-specific T cells are induced and APCs mediate the generation of T lymphocytes. We observe that after s.c. injection, MHC class II(+) cells infiltrate injection sites and are loaded with large amounts of antigen in vivo under the influence of poly-L-arginine. Consequently, numerous antigen-charged APCs can be detected in draining lymph nodes of vaccinated animals. Antigen-specific T cell responses induced are systemic and were readily detected more than 4 months after the last vaccination, the latest time point we measured. By contrast, even after repeat injections, we were consistently unable to detect antibody responses against poly-L-arginine, allowing this compound to be used for numerous booster injections. Clinical trials in cancer patients using poly-L-arginine as immunostimulant will be carried out in the near future.