Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles

Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (SNCA-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.</p

Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles

<div><p>Parkinson's disease (PD) is a progressive and currently incurable neurological disorder characterised by the loss of midbrain dopaminergic neurons and the accumulation of aggregated alpha-synuclein (a-syn). Oligomeric a-syn is proposed to play a central role in spreading protein aggregation in the brain with associated cellular toxicity contributing to a progressive neurological decline. For this reason, a-syn oligomers have attracted interest as therapeutic targets for neurodegenerative conditions such as PD and other alpha-synucleinopathies. In addition to strategies using small molecules, neutralisation of the toxic oligomers by antibodies represents an attractive and highly specific strategy for reducing disease progression. Emerging active immunisation approaches using vaccines are already being trialled to induce such antibodies. Here we propose a novel vaccine based on the RNA bacteriophage (Qbeta) virus-like particle conjugated with short peptides of human a-syn. High titres of antibodies were successfully and safely generated in wild-type and human a-syn over-expressing (<i>SNCA</i>-OVX) transgenic mice following vaccination. Antibodies from vaccine candidates targeting the C-terminal regions of a-syn were able to recognise Lewy bodies, the hallmark aggregates in human PD brains. Furthermore, antibodies specifically targeted oligomeric and aggregated a-syn as they exhibited 100 times greater affinity for oligomeric species over monomer a-syn proteins in solution. In the SNCA-OVX transgenic mice used, vaccination was, however, unable to confer significant changes to oligomeric a-syn bioburden. Similarly, there was no discernible effect of vaccine treatment on behavioural phenotype as compared to control groups. Thus, antibodies specific for oligomeric a-syn induced by vaccination were unable to treat symptoms of PD in this particular mouse model.</p></div

Effects of long-term immunisation with Qb-PD vaccines on a-syn aggregates.

<p>Male and female <i>SNCA</i>-OVX mice received 20 μg of Qb, Qb-PD1, Qb-PD3 or PBS every two weeks for a month, followed by monthly injections for 12 months (total duration of immunisation: for 13 months). (A-D) Immunofluorescence analysis was performed to detect a-syn aggregates with PLA in the substantia nigra, striatum, hippocampus and cerebellum. PLA puncta were quantified using ImageJ. (E) PLA puncta in 26–36 TH positive neurons were quantified and expressed as average mean of puncta per positive cell ± SEM (n = 3–4 mice per group), ANOVA followed by Dunn’s post hoc test. Scale bar 50 μm.</p

Assessment of behavioural effects of Qb-PD vaccines following long-term immunisation.

<p>Male and female <i>SNCA</i>-OVX mice received 20 μg of Qb, Qb-PD1, or Qb-PD3, or PBS every two weeks for a month, followed by monthly injections for 12 months (total duration of immunisation: 13 months). Parameters examined were (A) Rotarod fall latency, (B) spontaneous locomotor activity, (C) gait cadence, (D) forelimb stride length, (E) forelimb swing speed, (F) dry stool weight, (G) stool water content and (H) stool pellet number. Data are expressed as mean ± SEM (n = 13–16 mice per group).</p

Recognition of a-syn in human and mouse brain tissue.

<p>(A) Immunohistochemistry on paraffin-embedded brain tissue from a PD patient and control individual. Purified IgGs (1 mg/mL) used at 1:1000, commercial SYN211 antibody used at 1:2000. Region sampled, substantia nigra. Immunofluorescence on free-floating sections from the (B) striatum and (C) substantia nigra of 3 month old <i>SNCA</i>-OVX mice. Purified IgGs (1 mg/mL) used at 1:250, commercial BD a-syn antibody used at 1:500. Scale bars, 50 μm. (D) WB on 20 μg of striatum brain lysate from 3 month old mice. Samples from <i>Snca-/-</i> mice, mice over-expressing a-syn at moderate levels (line 21) and mice over-expressing a-syn with a two-fold increase (<i>SNCA</i>-OVX) were loaded (left-right). The MJFR1 antibody was used at 1:1000, while IgGs (1 mg/mL) from vaccinated mice were used at 1:500. Actin was used a loading control.</p

Competition ELISA to estimate relative affinity of vaccine-induced antibodies.

<p>Antigens in the form of peptides or recombinant monomer/oligomer protein preparations as described in the methods were coated on to microtitre plates and ELISA reading measured with fixed concentrations of serum IgG antibodies (at OD50 dilution) purified from pooled sera of vaccinated mice (n = 4), preincubated with serial dilutions of (A) free a-syn peptides (PD1, left; PD3, right); (B) free a-syn protein, either monomeric (left panels) or oligomeric (right panels) species, PD1 (top) and PD3 (bottom); and (C) liberated a-syn from haemolysed RBCs (orange line) or PBS negative control (blue line) for PD1 (top) and PD3 (bottom).</p

Assessment of a-syn levels following short-term immunisation.

<p>Male and female <i>SNCA</i>-OVX mice received 20 μg of Qb, Qb-PD1, or Qb-PD3, or PBS every two weeks for a month, followed by monthly injections for 1 or 2 months (total duration of immunisation: 2 or 3 months) and effects of Qb-PD vaccines on total a-syn protein levels in <i>SNCA</i>-OVX mice were examined. Striatum and midbrain lysates were prepared and 20 μg of protein was loaded per lane. Alpha-synuclein protein levels were measured by (A) ELISA, and WB following (B) SDS-PAGE and (C) native PAGE. Data are expressed as mean ± SEM (n = 3–4 mice per group) and were analysed using one-factor analysis of variance (ANOVA).</p

Age of mice and duration of immunisations for each experimental study group.

<p>Age of mice and duration of immunisations for each experimental study group.</p

Characterisation of a-syn oligomer preparations and recognition of these oligomers by IgGs from Qb-PD vaccinated mice.

<p>(A) Western blot (WB) of monomer and oligomer a-syn preparations reduced with (+) or without heating (-) probed with 4D6 a-syn antibody (1:2000). (B) A 100 μL sample of each preparation was passed through a Sephacryl S100-HR column, displaying over-layered chromatograms to compare UV (280nm) elution profiles. (C) Negative stained TEM of tangled oligomer preparation (scale bar, 100nm). (D) WB on 0.1 μg of full-length recombinant a-syn monomers (left lane) and a-syn oligomers prepared with 4-hydroxy-2-nonenal (HNE) (right lane). WB were probed using SYN211 a-syn antibody (1:5000) and IgGs (1:500) from vaccinated male and female <i>SNCA</i>-OVX mice. These vaccinated mice received 20 μg of Qb, Qb-PD1 or Qb-PD3 every two weeks for a month, followed by a monthly injection (total duration of immunisation: 2 months). Pre-immune refers to sera of mice collected prior to first immunisation at d0.</p

Qb VLP and PD vaccine preparation.

<p>(A) Qb positive fractions from anion exchange were pooled and concentrated prior to size exclusion chromatography (SEC) on 16/600 Sephacryl S500-HR column, elution monitored by UV at 280 nm (blue), 254 nm (red) and 220 nm (pink). (B) A sample from the major SEC peak (adjusted to 0.1 mg/mL) was negatively stained and viewed by TEM (scale bar, 100nm). (C) Coomassie stained SDS-PAGE of peptide conjugated VLP preparation of PD vaccines. Purified VLPs (lane 1), derivatised with SMPH (lane 2) and subsequent conjugation with peptides PD1 (lane 3), PD2 (lane 4) and PD3 (lane 5) (Precision Plus Protein Standards, Bio-Rad. Sizes in kDa as indicated). (D) Vaccine preparations loaded on native agarose gel were stained for nucleic acid with SYBR safe (left) and for protein with Coomassie (right). Purified VLPs (lane 1), derivatised with SMPH (lane 2), conjugated with peptides PD1 (lane 3) and PD3 (lane 4).</p