Now that a potential molecular target has been elucidated, is it realistic to think in terms of a therapeutic vaccine for Parkinson’s disease? Certainly say Markus Mandler and Achim Schneeberger, but there is a problem – how to avoid autoimmunity?     Parkinson’s disease (PD) is the second most common neurodegenerative disorder of the elderly (behind Alzheimer’s disease (AD)¹; in people aged >65, its prevalence ranges between 100 and 250 cases per 100,000 in Western countries and 1,700 per 100,000 in China. It was long considered a motor disease characterised by akinesia/bradykinesia, rigidity and rest tremor. Its non-motor symptoms (neuropsychiatric, gastrointestinal, autonomous symptoms) are now well established^2,3. PD treatments available to date primarily address the disease’s motor component. As they deliver symptomatic benefit only, they ultimately lose their clinical activity and fail³. Moreover, long-term use is associated with complications such as treatment induced dyskinesia. There are several areas of medical need. First, we lack a disease-modifying agent. Second, measures to combat treatment-induced dyskinesia are limited. Third, there are only a few therapeutic options for non-motor symptoms. Obviously, rational development of a disease-modifying therapeutic approach primarily requires understanding the underlying pathology. In the case of PD, there is mounting evidence for a causal and essential pathophysiological role of alpha-synuclein (aSyn). This field was pioneered by Spillantini and colleagues when they identified aSyn, by means of immunohistochemistry, to be a component of Lewy bodies⁴. Further arguments (see also Table 1) supporting a key role of aSyn in the pathogenesis of PD include the demonstration that certain dominantly inherited PD forms are caused by mutations in/duplications of the aSyn gene^5-9, the observation that aSyn represents the major component of the neuropathological signature lesions in patients (Lewy bodies and Lewy neurites)^10-12, the GWAS finding that certain aSyn gene variants contain the highest risk for sporadic PD^13-16, the demonstration that overexpression of human aSyn recapitulates certain features of the disease in experimental animals^17-21, and, finally, the fact that clinical symptoms and their progression correlate with the localisation and development of aSyn pathology^{11, 22}. The latter is further supported by studies demonstrating pathological aSyn to be transmitted from cell to cell in a prion-like propagation pathway²⁴. While all these studies suggest aSyn to be the toxic culprit, likely in the form of oligomers, downstream events such as mitochondrial dysfunction and oxidative stress are expected to mediate and even modulate its toxicity.

Beyond PD, aSyn was identified in hallmark lesions of other entities including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Together with further arguments supporting the assumption that aSyn plays a key pathological role in these conditions as well, this led to the definition of the group of neurodegenerative disorders designated as synucleopathies²⁵. In keeping with the above and further arguments, reduction of aSyn deposition and oligomerisation is expected to exhibit a disease-modifying effect. Masliah and colleagues were the first to test this hypothesis²⁶. They treated mice overexpressing the human aSyn gene with an aSyn-based vaccine. Mice developing high affinity Abs were found to have reduced amounts of aggregated aSyn both within their cell bodies and at the synapses. Experimental evidence provided suggests degradation of aSyn to occur via the lysosomal pathway. Importantly, the reduction of aSyn oligomers was found to correlate inversely with the extent of neurodegeneration. The above investigators obtained similar effects when using an aSyn-specific Ab in the sense of passive immunotherapy. While the above studies underline the value of aSyn-based immunotherapy, they do not allow for a full safety evaluation. Concerns are raised at two principal levels. One, using full length aSyn, a protein of 140 amino acids, would activate autoreactive T cells. Second, (complete) clearance of aSyn bears the risk of knocking-out its physiological function(s). Figure 1: Vaccination with PD-AFFITOPEs reduces cerebral aSyn levels in tg mice overexpressing human aSyn
Groups of transgenic mice (PDGF-aSyn) received 6 s.c. immunizations using a PD-AFFITOPE-vaccine or vehicle. Brain homogenates were analyzed for aSyn burden by Western blot and levels were quantified densitometrically. Asterisk denotes p<0.05[/caption] Both of the above concerns can be dealt with by the AFFITOME technology, which uses short peptides mimicking the target sequence²⁷. Key in the case of aSyn is to prevent the elicitation of antibodies reacting with other members of the synuclein family. This consists of three members – alpha, beta and gamma-synuclein – which do not only share extensive sequence homology but also a similar expression pattern. They are thought to act as chaperones thereby regulating vesicle transport and synaptic plasticity. Of note, aSyn knock-out mice are viable and show no overt neuronal phenotype indicating that aSyn function can be replaced by other synucleins. bSyn shows the highest overlap of expression with aSyn. It appears to counteract most of the toxic effects of aSyn. Its neuroprotective properties are in part mediated by AKT signalling. In addition, bSyn has been shown to prevent aggregation and oxidation of aSyn. Obviously, immunotherapeutic approaches need to respect these relations, in particular a vaccine targeting aSyn must not attack bSyn. In line with the above, the AFFiRiS PD drug development program focuses on AFFITOPEs eliciting Abs recognising aSyn while sparing bSyn. It delivered a series of candidates. These have been extensively tested in various transgenic preclinical models of synucleopathies²⁸. AFFITOPE vaccination resulted in high target-specific (aSyn) antibody titres in plasma, antibodies were able to cross the blood brain barrier as evidenced by different techniques (in situ staining and CSF analysis). Antibodies elicited were shown to primarily recognise aSyn aggregates. Induction of these antibodies was associated with a lowering of cerebral aSyn burden, especially of its oligomeric form (see Fig 1). This was accompanied by a reduction of the degeneration of tyrosine hydroxylase expressing nerve cells. Most importantly, successfully immunised mice were also shown to exhibit improvements in motor as well as memory deficits (depending on the model system used). Studies to the mode of action indicate that clearance of aSyn involves the activation of microglia in the context of a milieu of anti-inflammatory cytokines²⁸. Regarding safety, as expected, these peptide-protein conjugate vaccines do only elicit carrier-specific but not target-specific T cells. Thus, they avoid cellular autoimmunity. At the time points investigated, we do not have evidence for overt microgliosis, astrocytosis or a potential pathological inflammatory process. Together, these results demonstrate aSyn AFFITOPE vaccines to be safe on preclinical grounds and to exhibit disease-modifying properties. The term “disease modification” denotes an intervention that brings about a beneficial change in the pathology underlying a given disease. During the last decade, several treatment modalities have been analysed in for their neuroprotective potential in PD and other synucleopathies. These include classical PD drugs such as MAO B inhibitors but also novel approaches like gene therapy (e.g., adenovirus-mediated transfer of the nerve growth factor neurturin), intraventricular application of growth factors (e.g., PDGF) as well as stem cell transplantation. All of them have so far failed. One explanation may be that all of them targeted downstream events in the pathological cascade. PD01A is the first aSyn-addressing drug/vaccine introduced into phase I clinical testing (clinicaltrials.gov: NCT01568099). Primary goal of this study is the assessment of the safety/tolerability of repeated immunisations with PD01A. This is based on the assumption that a disease-modifying effect would require a long-lasting immune response. Regarding efficacy endpoints, the situation is, for obvious reasons, characterised by a lack of knowledge with regard to clinical and biological endpoints. As a result, basic principles need to be followed. Some of them are already implemented in an exploratory manner into the phase I study assessing PD01A. Common sense and experience from the Alzheimer immunotherapy field suggest the effort of disease modification to be most promising, if not solely possible, if one intervenes early during the course of the disease. This obviously is associated with a decreasing diagnostic accuracy. Other principles include the following: disease modification is a process (i) the effects of which add up over time and (ii) it should positively affect all domains affected by the disease. This requires one to assess a broad range of symptoms of the disease over a relevant period of time. Accumulating evidence supports a key role of aSyn in the pathophysiology of entities such as PD, MSA and DLB. Preclinical studies demonstrate the possibility to modify aSyn pathology driven by transgenic expression of the human aSyn gene, thus models of synucleopathies. PD01A is the first aSyn-targeting drug/vaccine that has entered the phase of clinical development. Progress in the field will also depend on improving the methodology used to assess clinical symptoms as well as on a better understanding of the disease process and associated biological markers. References

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Authors Markus Mandler and Achim Schneeberger of AFFiRiS Acknowledgements: This work was supported by grants from Austrian Science Promotion Agency (FFG), Vienna, Austria and the Michael J. Fox Foundation, New York, USA.