martin bachmann, immunologie bern use of virus-like ... · 1 regulation of anti-viral b cell...
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1
Regulation of anti-viral B cell responses
Use of virus-like particles for therapeutic vaccination
against addiction and other chronic diseases
Neue Möglichkeiten der Immuntherapie
Martin Bachmann, Immunologie Bern
History of Vaccinology
live virulent live attenuated/ inactivated recombinant
Variolation (pre-1700) Vaccinia (1796)
HBV subunit (1982) Conjugate (Hib) (1987) Genetically-modified pertussis (1995)
HPV
Rabies (1885) Cholera (1896)
Allergy (1911) Tuberculosis (1921)
Diphtheria (1923) Tetanus (1926) Yellow Fever (1935) Polio (1952) Measles (1958)
Mumps (1967) Rubella (1970)
Europe: 1800 (India, China: 1000)
2000 1900
year
specificity and safety
Source: Bachmann and Dyer, Nature Reviews Drug Discovery, 3, 81 – 88 (2004), WHO
2
3
Risk Factors in the 21st Century Growing Burden of Ageing Societies
4
• Hypertension Ø 40%-60% Patienten non-compliant within 1 year
• Hypercholesterinema Ø 45% non-compliant within 1 year Ø 60% non-compliant within 18 months
• Asthma Ø 50% non-compliant within year
Sources: Am J Med 1997; 102:43-49. Am J Man Care 1997; 3:s12-17. Ann Allergy Asthma Immunol 1997;79:177-186.
Poor compliace is a general problem
Risk Factors in the 21st Century
5
Drugs treating risk factors have to be:
• Conveniant, to maximize • Compliance
à Vaccines are conveniant since they have a long- lasting effect
The Treatment of Risk Factors
Risk Factors in the 21st Century
6
ACE inhibitors
(Renin inhibitors)
AIIR blocker
Modulate the Action of Angiotensin II
Hypertension Vaccine
7
Hypertension
Remaining Problems
Sources: NHS, (2004); AJH 17: 347-335 (2004)
• Compliance: An estimated 50-80% do not take all of the prescribed medication
• Morning pressor surge: Pharmacokinetic profile of current inhibitors of the RAS may limit efficacy in early morning hours
Vaccine targeting angiotensin II may address these two issues, due to long-lived antibody responses
8
Organization: low absent high
Antibody response + - +++
- - Induction of auto-antibodies +++
Science 262, 1448-1451
Antigen Organization drives B cell responses Repeptitivenes: a pathogen associated geometric pattern
9
Why is epitope reptitiveness so important
Our immune system exploits structural features for the discrimination of self and foreign.
Viruses cannot help but have to express highly repetitive and highly ordered arrays of antigens on their surface
Viruses have small genomes and therefore consist of a small number of proteins.
Role of epitope densitiy and organisation
à Antigen Organisation is a geometric PAMP (Pathogen associated molecular pattern)
Bachmann & Dyer Nat Rev Drug Discov. 2004
Active immunization: Mechanism
10
Bachmann & Dyer Nat Rev Drug Discov. 2004
No IgG antibodies in the absence of T help
T help provided by the carrier is required for antibody production à Hence no boosting of the response by endogenous
cytokine
11
Carrier-specific help bypasses Th cell tolerance
Bachmann & Dyer Nat Rev Drug Discov. 2004 12
Vaccine Design
Carrier
Linker (SMPH)
Antigen
NN
O
NO
O
O
O O
OCys
Lys
13
• Non-replicating • Contains RNA as natural TLR7/8 ligand • Very stable • Economic production in bacteria • 2 g/l bacterial culture of GMP grade material
Bachmann&Jennnings Nature reviews Immunology 10:787-796
14
10
100
1000
10000
Peptide Peptide Peptide- Protein Carrier
Peptide- VLP
Ant
ibod
y tit
er (
OD
50)
Adjuvants none Alum Alum none
Factor 100
Mouse
10
100
1000
10000
100000
50µg i.m.
50µg s.c.
10µg i.m.
10µg s.c.
Human
High Antibody Titers in Mice and Men
Ant
ibod
y tit
er (
End
poin
t)
J Allergy Clin Immunol.117:1470
15
Vaccine Design
Vaccine Design
CYT006-AngQb Qbeta virus-like particle
CGGDRVYIHPF
30 nm
Angiotensin II
16
Strong antibody responses against Angiotensin II Efficient Antibody Induction in SHR Rats
0
10'000
20'000
30'000
40'000
d7 d14 d21 d28
CYT006-AngQb E
LIS
A tit
er (O
D50
%)
Days after immunization
J Hypertens 25:63-72
17
SHR Rats: BP Reading by Telemetry
Reduction of blood pressure in rats
*
150
160
170
180
190
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Days
Sys
tolic
BP
(mm
Hg)
0
4000
8000 Anti A
ng II titer (OD
50%)
Titer CYT006-AngQb BP VLP BP CYT006-AngQb
p < 0.05
J Hypertens 25:63-72
18
Indication Mild to moderate hypertension (systolic 140–179mmHg; diastolic 90–109mmHg)
Design Double-blind, randomized, placebo-controlled,
sequential two-dose comparison study in 72 patients Study period: 4 months plus 8 months safety follow-up
Endpoints Safety, tolerability, and exploratory efficacy
(change from baseline blood pressure)
Study Outline (1) A Placebo-controlled Phase IIa Clinical Trial
19
Study Outline (2) Two Dose Levels vs. Placebo
N=24 100µg AngQb
placebo N=12
0 1 2 3 4
safety follow up
12 months
N=24 300µg AngQb
placebo N=12 safety follow up 24h ambulatory
blood pressure measurement
Injection
The Lancet, 371 (2008) 821-827
The Lancet, 371 (2008) 821-827
20
Antibody Responses in Study 01
0
4000
8000
0 4 8 12 16 20 24 28 32 36 40 44 48 weeks
ELI
SA
titer
300 µg AngQb 100 µg AngQb Placebo
6 14 10
Evidence for Affinity Maturation
21
Mean Change of ABP: 300 µg vs. Placebo
Day-time blood pressure
Day-time Blood Pressure
-9.0 / -4.0 mm Hg p=0.015 / p=0.064
at 8am -25 / -13 mm Hg p<0.0001 / p=0.0035
The Lancet, 371 (2008) 821-827
22
24 Hours Measurement
placebo (SBP/DBP), n=12 300µg AngQb (SBP/DBP), n=21
70 80 90
100 110 120 130 140 150 160 170
09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 00 01 02 03 04 05 06 07 08
Time (24-hour)
Am
bula
tory
blo
od p
ress
ure
(mm
Hg)
run-
in
The Lancet, 371 (2008) 821-827
23
Conclusion
• Immunization against angiotensin II can significantly reduce blood-pressure in hypertensive individuals • Regimen and dosing, however, needs optimization
Regulation of anti-viral B cell responses
Use of virus-like particles for therapeutic vaccination
against addiction and other chronic diseases
Vaccination against Parkinson`s disease Martin Bachmann, Jenner Institute, University of Oxford, UK
Parkinson’s Disease (PD)
Neurodegenerative disease Loss of dopaminergic neurons Leading to motor control disorder Characterised by Lewy bodies = protein aggregates in
the brain
Mis-folded / aggregated disease-specific proteins common feature of neurodegenerative diseases
PrP, Kreuzfeld-Jakob disease, etc Amyloid-β (Aβ), tau - Alzheimer’s disease (AD) α-syn – Parkinson’s disease (PD) Huntigtons, ALS 25
α-Synuclein
§ α-synuclein is expressed in various tissues and adopts a monomeric but
largely unfolded structure (J Biol Chem. 2012 May 4;287(19):15345-64.).
§ α-synuclein undergoes heavy posttranslational modification, including
phosphorylation, nitration and sometimes aggregation (Nat Rev Neurosci.
2013 Jan;14(1):38-48)
§ Mild overexpression (2-fold) but also single point mutations may cause
familial Parkinson`s disease (Lancet 364(9440):1167–1169.; Science 276:
2045)
§ Aggregated α-synuclein is thought to be central for Parkinson`s disease
development
26
Parkinson’s Disease (PD)
REVIEWdoi:10.1038/nature12481
Self-propagation of pathogenic proteinaggregates in neurodegenerative diseasesMathias Jucker1,2 & Lary C. Walker3,4
For several decades scientists have speculated that the key to understanding age-related neurodegenerative disorders may befound in the unusual biology of the prion diseases. Recently, owing largely to the advent of new disease models, this hypothesishas gained experimental momentum. In a remarkable variety of diseases, specific proteins have been found to misfold andaggregate into seeds that structurally corrupt like proteins, causing them to aggregate and form pathogenic assemblies rangingfrom small oligomers to large masses of amyloid. Proteinaceous seeds can therefore serve as self-propagating agents for theinstigation and progression of disease. Alzheimer’s disease and other cerebral proteopathies seem to arise from the de novomisfolding and sustained corruption of endogenous proteins, whereas prion diseases can also be infectious in origin. However,the outcome in all cases is the functional compromise of the nervous system, because the aggregated proteins gain a toxicfunction and/or lose their normal function. As a unifying pathogenic principle, the prion paradigm suggests broadly relevanttherapeutic directions for a large class of currently intractable diseases.
P roteins are essential to cellular metabolism and communication,and they form the framework on which cells and tissues are built.To undertake these roles, most proteins fold into a specific, three-
dimensional architecture that is largely determined by their distinctivesequences of amino acids. Others have a degree of structural flexibility thatenables them to tailor their shape to the task at hand1,2. For proteins, then, asfor the rest of biology, structure governs function. Hence, it is critical for cellsto maintain an efficient quality-control system that ensures the properproduction, folding and elimination of proteins3,4. When a protein misfoldsand evades normal clearance pathways, a pathogenic process can ensue in
which the protein aggregates progressively into intracellular and/or extra-cellular deposits. The consequence is a diverse group of disorders, each ofwhich entails the aggregation of particular proteins in characteristic patternsand locations (Fig. 1)5–8. New insights into the ontogeny of these proteo-pathies are beginning to emerge from the unusual properties of the prion,arguably one of the most provocative molecules in the annals of medicine.
The prion paradigmPrions (‘proteinaceous infectious particles’) are unconventional infec-tious agents consisting of misfolded prion protein (PrP) molecules; in
1Department of Cellular Neurology, Hertie Institute for Clinical Brain Research, University of Tubingen, D-72076 Tubingen, Germany. 2DZNE, German Center for Neurodegenerative Diseases, D-72076Tubingen, Germany. 3Yerkes National Primate Research Center, Emory University, Atlanta, Georgia 30329, USA. 4Department of Neurology, Emory University, Atlanta, Georgia 30322, USA.
a
b
c
d
e
t
t
t
t
f
g
h
Figure 1 | Commonalities among age-relatedneurodegenerative diseases. The depositedproteins adopt an amyloid conformation and showprion-like self-propagation and spreading inexperimental settings, consistent with theprogressive appearance of the lesions in the humandiseases. a, Amyloid-b deposits (senile plaques) inthe neocortex of a patient with Alzheimer’s disease.b, Tau inclusion as a neurofibrillary tangle in aneocortical neuron of a patient with Alzheimer’sdisease. c, a-Synuclein inclusion (Lewy body) in aneocortical neuron from a patient with Parkinson’sdisease/Lewy body dementia. d, TDP-43 inclusionin a motoneuron of the spinal cord from a patientwith amyotrophic lateral sclerosis. Scale bars are50mm in a and 20mm in b–d. e–h, Characteristicprogression of specific proteinaceous lesions inneurodegenerative diseases over time (t, blackarrows), inferred from post-mortem analyses ofbrains. Amyloid-b deposits and tau inclusions inbrains of patients with Alzheimer’s disease (e and f),a-synuclein inclusions in brains of patients withParkinson’s disease (g), and TDP-43 inclusions inbrains of patients with amyotrophic lateral sclerosis(h). Three stages are shown for each disease, withwhite arrows indicating the putative spread of thelesions (for details see refs 5–8). Panels e and f arereproduced, with permission, from ref. 61.
5 S E P T E M B E R 2 0 1 3 | V O L 5 0 1 | N A T U R E | 4 5
Macmillan Publishers Limited. All rights reserved©2013
M Jucker and LC Walker, (2013) Self-propagation of pathogenic protein aggregates in neurodegenerative diseases Nature; 501(7465):45-51
27
Antibodies may stop propagation
Small aggregates / oligomers neutralised by antibodies
Protection of degenerative pathology
Hope for approach to stop progression and spread of proteo-pathological disorders
28
Antibodies protect against PD in mouse model
Passive Immunization Reduces Behavioral andNeuropathological Deficits in an Alpha-SynucleinTransgenic Model of Lewy Body DiseaseEliezer Masliah1,2*, Edward Rockenstein1, Michael Mante1, Leslie Crews2, Brian Spencer1, Anthony
Adame1, Christina Patrick1, Margarita Trejo1, Kiren Ubhi1, Troy T. Rohn3, Sarah Mueller-Steiner4, Peter
Seubert4, Robin Barbour4, Lisa McConlogue4, Manuel Buttini4, Dora Games4, Dale Schenk4
1 Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America, 2 Department of Pathology, University of California San
Diego, La Jolla, California, United States of America, 3 Department of Biology, Boise State University, Boise, Idaho, United States of America, 4 ELAN Pharmaceuticals, South
San Francisco, California, United States of America
Abstract
Dementia with Lewy bodies (DLB) and Parkinson’s Disease (PD) are common causes of motor and cognitive deficits and areassociated with the abnormal accumulation of alpha-synuclein (a-syn). This study investigated whether passiveimmunization with a novel monoclonal a-syn antibody (9E4) against the C-terminus (CT) of a-syn was able to cross intothe CNS and ameliorate the deficits associated with a-syn accumulation. In this study we demonstrate that 9E4 was effectiveat reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved a-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4traffics into the CNS, binds to cells that display a-syn accumulation and promotes a-syn clearance via the lysosomalpathway. These results suggest that passive immunization with monoclonal antibodies against the CT of a-syn may be oftherapeutic relevance in patients with PD and DLB.
Citation: Masliah E, Rockenstein E, Mante M, Crews L, Spencer B, et al. (2011) Passive Immunization Reduces Behavioral and Neuropathological Deficits in anAlpha-Synuclein Transgenic Model of Lewy Body Disease. PLoS ONE 6(4): e19338. doi:10.1371/journal.pone.0019338
Editor: Grainne M. McAlonan, The University of Hong Kong, Hong Kong
Received December 22, 2010; Accepted March 28, 2011; Published April 29, 2011
Copyright: ! 2011 Masliah et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health (NIH) grants AG 11385, AG 18840, AG 022074 and NS 044233 and by ELAN Pharmaceuticals. NIHhad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sarah Mueller-Steiner, Peter Seubert, RobinBarbour, Lisa McConlogue, Manuel Buttini, Dora Games and Dale Schenk are employed by ELAN Pharmaceuticals, who, in collaboration with the group at UCSD,were intellectually involved in the conception and execution of the in vitro and in vivo passive immunization experiments.
Competing Interests: The authors have declared the following conflict of interest: Sarah Mueller-Steiner, Peter Seubert, Robin Barbour, Lisa McConlogue,Manuel Buttini, Dora Games and Dale Schenk are employed by ELAN Pharmaceuticals. There are no patents, products in development or marketed products todeclare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: [email protected]
Introduction
Neurodegenerative conditions with accumulation of a-synuclein(a-syn) are common causes of dementia and movement disordersin the aging population. Disorders where the clinical andpathological features of Alzheimer’s Disease (AD) and Parkinson’sDisease (PD) overlap are known as Lewy body disease (LBD) [1].a-Syn is a natively unfolded protein [2] found at the presynaptic
terminal [3] and may play a role in synaptic plasticity [4].Abnormal a-syn accumulation in synaptic terminals and axonsplays an important role in LBD [5,6,7,8]. Recent work hassuggested that a-syn oligomers rather than fibrils might be theneurotoxic species [9,10].
While in rare familial cases mutations in a-syn might contributeto oligomerization [11], it is unclear what triggers a-synaggregation in sporadic forms of LBD. Alterations in a-synsynthesis, aggregation or clearance have been proposed to impactthe formation of toxic oligomers [12,13,14]. Therefore, strategiesdirected at promoting the clearance of oligomers may be oftherapeutic value for LBD. Previous studies have used genetherapy targeting selective regions to increase a-syn clearance viaautophagy or by reducing a-syn synthesis [12,15].
However, neurodegenerative processes in LBD are morewidespread than originally suspected [16] therefore there is aneed for therapeutic approaches that target toxic a-syn in multipleneuronal populations simultaneously. For this reason we began toexplore an immunotherapy approach for LBD and havepreviously shown that active immunization with recombinant a-syn ameliorates a-syn related synaptic pathology in a transgenic(tg) mouse model of PD [17]. Previous studies have shown thatintracellular antibodies (intrabodies) can inhibit a-syn aggregation[18,19] and that copolymer-1 immunotherapy reduces neurode-generation in a PD model [20].
The mechanisms through which a-syn immunotherapy mightwork are unclear given that native a-syn is cytoplasmic. However,it is possible that antibodies may recognize abnormal a-synaccumulating in the neuronal plasma membrane [10,17,21,22] orsecreted forms of a-syn. In support of this possibility, studies haveshown that oligomerized a-syn is secreted in vitro [23] and in vivo[24] via exocytosis, contributing to the propagation of thesynucleinopathy. Moreover, a-syn is present in the cerebrospinalfluid of a-syn tg mice and in patients with LBD [25,26].
This study examined whether passive immunization with anantibody against the C-terminus (CT) of a-syn (hereafter referred
PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19338
Passive Immunization Reduces Behavioral andNeuropathological Deficits in an Alpha-SynucleinTransgenic Model of Lewy Body DiseaseEliezer Masliah1,2*, Edward Rockenstein1, Michael Mante1, Leslie Crews2, Brian Spencer1, Anthony
Adame1, Christina Patrick1, Margarita Trejo1, Kiren Ubhi1, Troy T. Rohn3, Sarah Mueller-Steiner4, Peter
Seubert4, Robin Barbour4, Lisa McConlogue4, Manuel Buttini4, Dora Games4, Dale Schenk4
1 Department of Neurosciences, University of California San Diego, La Jolla, California, United States of America, 2 Department of Pathology, University of California San
Diego, La Jolla, California, United States of America, 3 Department of Biology, Boise State University, Boise, Idaho, United States of America, 4 ELAN Pharmaceuticals, South
San Francisco, California, United States of America
Abstract
Dementia with Lewy bodies (DLB) and Parkinson’s Disease (PD) are common causes of motor and cognitive deficits and areassociated with the abnormal accumulation of alpha-synuclein (a-syn). This study investigated whether passiveimmunization with a novel monoclonal a-syn antibody (9E4) against the C-terminus (CT) of a-syn was able to cross intothe CNS and ameliorate the deficits associated with a-syn accumulation. In this study we demonstrate that 9E4 was effectiveat reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved a-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4traffics into the CNS, binds to cells that display a-syn accumulation and promotes a-syn clearance via the lysosomalpathway. These results suggest that passive immunization with monoclonal antibodies against the CT of a-syn may be oftherapeutic relevance in patients with PD and DLB.
Citation: Masliah E, Rockenstein E, Mante M, Crews L, Spencer B, et al. (2011) Passive Immunization Reduces Behavioral and Neuropathological Deficits in anAlpha-Synuclein Transgenic Model of Lewy Body Disease. PLoS ONE 6(4): e19338. doi:10.1371/journal.pone.0019338
Editor: Grainne M. McAlonan, The University of Hong Kong, Hong Kong
Received December 22, 2010; Accepted March 28, 2011; Published April 29, 2011
Copyright: ! 2011 Masliah et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by National Institutes of Health (NIH) grants AG 11385, AG 18840, AG 022074 and NS 044233 and by ELAN Pharmaceuticals. NIHhad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sarah Mueller-Steiner, Peter Seubert, RobinBarbour, Lisa McConlogue, Manuel Buttini, Dora Games and Dale Schenk are employed by ELAN Pharmaceuticals, who, in collaboration with the group at UCSD,were intellectually involved in the conception and execution of the in vitro and in vivo passive immunization experiments.
Competing Interests: The authors have declared the following conflict of interest: Sarah Mueller-Steiner, Peter Seubert, Robin Barbour, Lisa McConlogue,Manuel Buttini, Dora Games and Dale Schenk are employed by ELAN Pharmaceuticals. There are no patents, products in development or marketed products todeclare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
* E-mail: [email protected]
Introduction
Neurodegenerative conditions with accumulation of a-synuclein(a-syn) are common causes of dementia and movement disordersin the aging population. Disorders where the clinical andpathological features of Alzheimer’s Disease (AD) and Parkinson’sDisease (PD) overlap are known as Lewy body disease (LBD) [1].a-Syn is a natively unfolded protein [2] found at the presynaptic
terminal [3] and may play a role in synaptic plasticity [4].Abnormal a-syn accumulation in synaptic terminals and axonsplays an important role in LBD [5,6,7,8]. Recent work hassuggested that a-syn oligomers rather than fibrils might be theneurotoxic species [9,10].
While in rare familial cases mutations in a-syn might contributeto oligomerization [11], it is unclear what triggers a-synaggregation in sporadic forms of LBD. Alterations in a-synsynthesis, aggregation or clearance have been proposed to impactthe formation of toxic oligomers [12,13,14]. Therefore, strategiesdirected at promoting the clearance of oligomers may be oftherapeutic value for LBD. Previous studies have used genetherapy targeting selective regions to increase a-syn clearance viaautophagy or by reducing a-syn synthesis [12,15].
However, neurodegenerative processes in LBD are morewidespread than originally suspected [16] therefore there is aneed for therapeutic approaches that target toxic a-syn in multipleneuronal populations simultaneously. For this reason we began toexplore an immunotherapy approach for LBD and havepreviously shown that active immunization with recombinant a-syn ameliorates a-syn related synaptic pathology in a transgenic(tg) mouse model of PD [17]. Previous studies have shown thatintracellular antibodies (intrabodies) can inhibit a-syn aggregation[18,19] and that copolymer-1 immunotherapy reduces neurode-generation in a PD model [20].
The mechanisms through which a-syn immunotherapy mightwork are unclear given that native a-syn is cytoplasmic. However,it is possible that antibodies may recognize abnormal a-synaccumulating in the neuronal plasma membrane [10,17,21,22] orsecreted forms of a-syn. In support of this possibility, studies haveshown that oligomerized a-syn is secreted in vitro [23] and in vivo[24] via exocytosis, contributing to the propagation of thesynucleinopathy. Moreover, a-syn is present in the cerebrospinalfluid of a-syn tg mice and in patients with LBD [25,26].
This study examined whether passive immunization with anantibody against the C-terminus (CT) of a-syn (hereafter referred
PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19338
29
30
Active versus passive vaccination
Drug Discov Today. 2006 Nov;11(21-22):1028-33 Nat Rev Drug Discov. 2004 Jan;3(1):81-8 Vaccine. 2013 Apr 3;31(14):1777-84
• mAb therapy is highly effective, but à cost-intensive à many patients develop anti-antibody responses which neutralize the therapeutic potential of mAbs • Increasingly older populations pose a pharmacoeconomic threat to health- care systems resulting in downward pressure on drug-costs • Innovative and cost-effective therapies are needed • Vaccination addresses these issues
31
Goal
1) Induction of antibodies recognizing oligomeric and
aggregated α-synuclein
2) Oligomers should be recognized preferentially over
monomeric α-synuclein (à even though native α-synuclein
is intracellular and not obviously accessible to antibodies).
3) Specific T cells should be avoided (see ELAN and their AD
vaccine) à no strong adjuvants and peptide epitopes
Vaccine Design
Carrier
Linker (SMPH)
Antigen
NN
O
NO
O
O
O O
OCys
Lys
32
• Non-replicating • Contains RNA as natural TLR7/8 ligand • Very stable • Economic production in bacteria • 2 g/l bacterial culture of GMP grade material
Bachmann&Jennnings Nature reviews Immunology 10:787-796
Aβ1-6 DAEFRHGGC
AβQb
6
16
25
32
47 62 83
175
3 2 1
Epitopes per Qβ monomer
QβAβ1-6 Qβ
Aβ1-42 DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA
CAD106: A Vaccine for Alzheimer`s now in Phase III based on the same principle
33
- 3 peptides chosen for vaccine design: MDVFMKGL, KNEEGAPQ, EGYQDYEPEA
- Sequence comprised between 8-10 AA à essentially no T cell epitopes
- C-term and N-term of proteins generally accessible in aggregates
Amphipathic region Acidic tail NAC domain
1 61 95 140 N-
terminal C-
terminal
N-terminal mAb target C-terminal
Vaccine-Design: Epitope
Figure 2. Peptide-specific antibody responses induced by the three vaccines and a biosimilar of CAD106 (a vaccine against Alzheimer’s disease currently developed by Novartis) (n=4 mice/group).
Good response with all peptides
35
Amphipathic region Acidic tail NAC domain
1 61 95 140 N-
terminal C-
terminal
N-terminal mAb target C-terminal
IgG from immunised mice (VLP-‐pep6de 3) day 70
Figure 4. Immunohistochemistry on paraffin-embedded post-mortem brain tissue from a PD patient, Braak stage 4. Purified IgGs (1 mg/mL) used at 1:1000. Region sampled, substantia nigra. Scale bar, 50 µm. Lewy bodies (white arrows), Lewy neurites (black
arrows), Pale body (white arrowhead), intracellular aggregation (white dashed arrows).
Recognition of aggregated α-synuclein (human brain tissue)
36
IgG from immunised mice (VLP-‐pep6de 1) day 70 IgG from immunised mice (VLP-‐pep6de 2) day 70
Figure 3. Immunohistochemistry on paraffin-embedded post-mortem brain tissue from a PD patient, Braak stage 4. Purified IgGs (1 mg/mL) used at 1:1000. Region sampled, substantia nigra. Scale bar, 50 µm. Lewy bodies (white arrows), intracellular aggregation
(white dashed arrows).
Recognition of aggregated α-synuclein (human brain tissue)
37
Mouse Model of Parkinson`s disease progression of Parkinson's in patients. We will use this state-of-the art model to test the therapeutic efficacy of our novel vaccination approach targeting α-synuclein.
Figure 1. Characterisation of the SNCA-OVX mouse model. SNCA-OVX mice show expression of α-synuclein in TH+ve neurons of the SNc (A), have twice the level of endogenous α-synuclein expression (B), and lose 30% of SNc neurons at 18 months of age compared to the control transgenic line, expressing low levels of α-synuclein (hα−syn) (C). (D) SNCA-OVX mice exhibit reduced evoked release of dopamine from 3 months of age in the dorsal striatum. (E) At 18 months of age SNCA-OVX mice have a decreased ability to remain on the accelerating rotarod. (F) SNCA-OVX mice at 18 months of age take longer to traverse a narrow balance beam and show a greater number of foot-slips. 3) Workplan The setup of the workplan is summarized in Fig. 2.
Fig 2 Workflow of the program As done previously for a vaccine against Alzheimer`s disease, the PI (MFB) will design vaccines based on selected α-synuclein-derived peptides and one protein-based vaccine. Normal and α-synuclein-transgenic mice will be immunized and the induced antibodies will
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hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
A' C'
34 mth 12 mth 18 mth
Snca (/)
SNCA OVX
0.5 !"
0.5 s
Snca (/)
SNCA OVX
0.5 !"
0.5 s
0.5 !"
0.5 s
*** ** ***
A B C
Figure 3
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0.5 s
)*+,!"
3 mth
Snca (/)
#$%&'(
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18 mth
20 (A
700 mV +1300 mV
CPu NAc
CPu NAc
D
CPu
NAc
0.5 s
)*+,!"
**
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*
Snca (/)
SNCA OVX
)*+,!"
0.5 s
CPu NAc0
20
40
60
80
100
120
140
Regio(
[DA] (% of Snca/)
CPu NAc0
20
40
60
80
100
120
140
Regio(
CPu NAc0
20
40
60
80
100
120
140
Regio(
CPu NAc0
20
40
60
80
100
120
140
Regio(
CPu NAc0
20
40
60
80
100
120
140
Regio(
**
[DA] (% of Snca/)
[DA] (% of Snca/)
[DA] (% of Snca/)
[DA] (% of Snca/)
G H I
J K
CPu NAc CPu NAc
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Stim
Stim
D'
Snca-/-
A
C D
E
Figure 2
F
B
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
B'
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
3'months'
18'months'
E'
Snca-/-
A
C D
E
Figure 2
F
B
Traverse'
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Foot'slips'Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Vaccine'(Design(
An-body'specificity(Tes-ng(
Tes$ng'in'murine'model'
Early'process'development'
progression of Parkinson's in patients. We will use this state-of-the art model to test the therapeutic efficacy of our novel vaccination approach targeting α-synuclein.
Figure 1. Characterisation of the SNCA-OVX mouse model. SNCA-OVX mice show expression of α-synuclein in TH+ve neurons of the SNc (A), have twice the level of endogenous α-synuclein expression (B), and lose 30% of SNc neurons at 18 months of age compared to the control transgenic line, expressing low levels of α-synuclein (hα−syn) (C). (D) SNCA-OVX mice exhibit reduced evoked release of dopamine from 3 months of age in the dorsal striatum. (E) At 18 months of age SNCA-OVX mice have a decreased ability to remain on the accelerating rotarod. (F) SNCA-OVX mice at 18 months of age take longer to traverse a narrow balance beam and show a greater number of foot-slips. 3) Workplan The setup of the workplan is summarized in Fig. 2.
Fig 2 Workflow of the program As done previously for a vaccine against Alzheimer`s disease, the PI (MFB) will design vaccines based on selected α-synuclein-derived peptides and one protein-based vaccine. Normal and α-synuclein-transgenic mice will be immunized and the induced antibodies will
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Snca (/)
SNCA OVX
0.5 !"
0.5 s
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0.5 !"
0.5 s
0.5 !"
0.5 s
*** ** ***
A B C
Figure 3
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0.5 s
)*+,!"
3 mth
Snca (/)
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E F
18 mth
20 (A
700 mV +1300 mV
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CPu NAc
D
CPu
NAc
0.5 s
)*+,!"
**
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*
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SNCA OVX
)*+,!"
0.5 s
CPu NAc0
20
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Regio(
[DA] (% of Snca/)
CPu NAc0
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Regio(
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Regio(
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Regio(
CPu NAc0
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Regio(
**
[DA] (% of Snca/)
[DA] (% of Snca/)
[DA] (% of Snca/)
[DA] (% of Snca/)
G H I
J K
CPu NAc CPu NAc
CPu NAc
Stim
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Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
A' C'
34 mth 12 mth 18 mth
Snca (/)
SNCA OVX
0.5 !"
0.5 s
Snca (/)
SNCA OVX
0.5 !"
0.5 s
0.5 !"
0.5 s
*** ** ***
A B C
Figure 3
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#$%&'(
0.5 s
)*+,!"
3 mth
Snca (/)
#$%&'(
E F
18 mth
20 (A
700 mV +1300 mV
CPu NAc
CPu NAc
D
CPu
NAc
0.5 s
)*+,!"
**
*
*
Snca (/)
SNCA OVX
)*+,!"
0.5 s
CPu NAc0
20
40
60
80
100
120
140
Regio(
[DA] (% of Snca/)
CPu NAc0
20
40
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Regio(
CPu NAc0
20
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Regio(
CPu NAc0
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Regio(
CPu NAc0
20
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Regio(
**
[DA] (% of Snca/)
[DA] (% of Snca/)
[DA] (% of Snca/)
[DA] (% of Snca/)
G H I
J K
CPu NAc CPu NAc
CPu NAc
Stim
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D'
Snca-/-
A
C D
E
Figure 2
F
B
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E 3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
B'
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Figure 1
B
D
α‐syn
hα‐syn SNCA‐OVX Snca‐/‐ C57Bl6
A
C
E
3 m
on
ths
hα-syn SNCA-OVX Snca-/-
18 m
on
ths
hα-syn SNCA-OVX Snca-/-
F
G H
‐ PK + PK LB509
Syn‐1
Snca‐/‐
SNCA‐OVX
CA3 sg
sp
SNpc
SNpr
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
α‐synuclein TH α‐synuclein/ TH
hα‐syn
Snca‐/‐
SNCA‐OVX
80m 80m 80m
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
3'months'
18'months'
E'
Snca-/-
A
C D
E
Figure 2
F
B
Traverse'
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Foot'slips'Snca-/-
A
C D
E
Figure 2
F
B
Snca-/-
A
C D
E
Figure 2
F
B
Vaccine'(Design(
An-body'specificity(Tes-ng(
Tes$ng'in'murine'model'
Early'process'development'
Janezic et al., PNAS, 2013 38
111-kb wild-type human SNCA locus (Fig. S1A). Two mouse lineswere generated: SNCA-OVX, which overexpresses human α-synto levels that model those associated with SNCA multiplications(10), and hα-syn, which expresses human α-syn at a low/moderatelevel as a control for expression of human α-syn. Both lines were
bred to a mouse α-syn-null (Snca−/−) pure C57/Bl6 background(11) to preclude confounding interactions with endogenous α-syn.Complete transgene integration was confirmed by exon PCRamplification of all six human SNCA exons (Fig. S1B). ForSNCA-OVX mice, a single site of integration was identified by
B
Dɲ-syn
hɲ-synSNCA-OVXSnca-/- C57/Bl6
A
C
E
3 m
onth
s
h -synSNCA-Snca-/-
18 m
onth
s
h -synSNCA-OVXOVX Snca-/-F
G H
ɲ-synuclein TH ɲ-synuclein/ TH
h ɲ-s
ynSnca
-/-
SNCA
-OVX
ɲ-synuclein TH ɲ-synuclein/ TH
hɲ-s
ynSnca
-/-
SNCA
-OVX
SNCA-OVX Snca-/-
+AC
+ PK
LB50
9
Syn-1+FA
20 m20 m
20 m
20 m 20 m
20 m
20 m
20 m
20 m
20 m20 m
Thioflavine S
PD control
20 m
Fig. 1. Characterization of α-syn transgenic mice. (A and B) Double immunofluorescence labeling for α-syn and TH confirms human α-syn transgene ex-pression in TH-immunoreactive dopamine neurons of the (A) SNc and (B) VTA in 3-mo-old SNCA-OVX and hα-syn animals. (Scale bars, 200 μm.) (C) Quanti-tative Western analysis of striatal α-syn expression in 3-mo-old SNCA-OVX, hα-syn, Snca−/−, and C57/Bl6 animals reveals that SNCA-OVX animals express thehuman wild-type SNCA transgene at 1.9-fold higher levels compared with endogenous mouse α-syn protein in C57/Bl6 animals. No α-syn expression wasobserved in Snca−/− control animals. One-way ANOVA with Bonferroni post hoc analysis; ****P < 0.0001, ***P < 0.001, n = 2–3. (D) Immunohistochemicalanalysis revealed somatic cytoplasmic α-syn immunostaining in cells of the SNc in 18-mo-old SNCA-OVX mice using the Syn-1 antibody with formic acid (FA)pretreatment. Similar structures were weakly labeled in 18-mo-old SNCA-OVX mice using the LB509 antibody autoclaved (AC) in citric buffer. This staining wasabolished when proteinase K (PK) antigen retrieval was applied instead. All of these immunohistochemical stainings were carried out together with controltissue (entorhinal cortex from a PD patient with dementia that shows prominent Lewy body and neuritic pathology). No “amyloid” pathology was detectedwith thioflavine S in either 18-mo-old SNCA-OVX or Snca−/−, whereas several Lewy bodies and dystrophic neurites were detected in the positive PD control.(Scale bars, 20 μm; magnification: all pictures, 400×.) (E and G) At 3 mo of age, stereological cell counting revealed no differences in the number of TH-immunoreactive neurons in the SNc of SNCA-OVX mice compared with hα-syn and Snca−/− mice. One-way ANOVA: no main effect of genotype: F < 1, P > 0.05,n = 5 per genotype. Data are expressed as the mean ± SEM. Representative images of TH-immunoreactivity in the SNc are shown. (Scale bar, 200 μm.) (F and H)Analysis of 18-mo-old animals revealed a 30% loss of TH-immunoreactive neurons in the SNc of SNCA-OVX mice compared with hα-syn mice. One-way ANOVAwith Bonferroni post hoc analysis: main effect of genotype: F(2,12) = 6.3, *P < 0.05, n = 5 per genotype. Data are expressed as the mean ± SEM. Representativeimages of TH immunoreactivity in the SNc are shown. (Scale bar, 200 μm.)
Janezic et al. PNAS | Published online September 30, 2013 | E4017
NEU
ROSC
IENCE
PNASPL
US
α-syn is recognized in tg-mouse model
39
40
Goal
1) Induction of antibodies recognizing oligomeric and
aggregated α-synuclein
2) Oligomers should be recognized preferentially over
monomeric α-synuclein (à even though native α-synuclein
is intracellular and not obviously accessible to antibodies).
3) Specific T cells should be avoided (see ELAN and their AD
vaccine) à no strong adjuvants and peptide epitopes
41
Soluble proteins versus aggregates
à Low affinity antibodies recognize aggregates but not soluble proteins
Bivalent Binding à „avidity“ Monovalent Binding à „affinity“
α-synuclein specific Abs have low affinity
Peptide 1 200 nM 190 nM Peptide 3 30 nM 35 nM
Day 14
42
Day 70
Affinity of immune sera
2. a. Dopamine neurotransmission (FCV)
2. b. Behavioural studies
Study 2
VLP-peptide (20 μg), s.c. administra0on (every 2 weeks for 1 month, then monthly)
0 21 Experimental day (month)
28 (1)
(2) 7 14
Age of animal 10 wks
(3) (4) (5) (6)
14 wks (3.5 mo.)
6 wks
(12) (18)
12 mo.
18 mo.
1. Biochemistry: assess α-synuclein protein burden (ELISA, WB) Study 1
Vaccination of young mice (6 week-old)
Controls: - SNCA-OVX mice: administration of non-coupled VLPs
Efficacy Experiment Ongoing
43
Acknowledgements
Cytos Biotechnology, Zürich Gunther Spohn Patrik Maurer Gary Jennings
Hypertension Robert Sabat, Berlin Frank Wagner, Berlin Jürg Nussberger, Lausanne
44
University of Oxford Aadil El-Turabi Marika Doucet Richard Wade-Martins
University Hospital Zürich Franziska Zabel Antonia Fettelschoss Thomas Kündig
BRSC Riga Paul Pumpens Andris Zeltins Andris Dishlers