The amount of brain-associated α-syn aggregates in sick mice was not significantly different regardless of their immunization status, whether vaccinated or not, or the route of challenge, intraperitoneal (Supplementary Fig. 6A) or intragastric (Supplementary Fig. 6B). In conclusion, administration of α-syn fibrils via injections into the peritoneum or the wall of the gastrointestinal tract seeded a progressive synucleinopathy in all challenged mice, regardless of their immune status. Within each group, all mice exhibited signs of neurological disease when comparable amounts of α-syn aggregates were present in the brain. This suggests that vaccination protected mice and enabled their longer survival by slowing down the accumulation of detrimental amounts of α-syn aggregates in the brain.

In this study, we modelled body-first PD by challenging fully vaccinated TgM83+/− mice with injections of synthetic α-syn fibrils either into the peritoneum or into the wall of the stomach and pylorus, which seeds α-syn aggregation in the enteric nervous system first.

For intraperitoneally challenged mice, immunization with all vaccine candidates, with the exception of α-SC8 fibrils (236 days, P = 0.063), significantly prolonged survival in comparison to unvaccinated control mice with a median survival of 223 days (Fig. 2B and Table 1). Survival was most prolonged, by 38%, in mice immunized with α-SC6 fibrils (307 days, P < 0.0001). It is noteworthy that vaccination with unmodified HET-s fibrils (261 days, P < 0.05) also significantly protected mice from disease upon challenge, with a slightly more pronounced effect than that observed with α-SC3 fibrils (249 days, P < 0.05). We observed the most effective protection of vaccination in mice challenged with α-syn fibrils via the intragastric route (Fig. 2C and Table 1). In comparison to unvaccinated mice with a median survival of 231 days, vaccination with α-SC8 fibrils (328.5 days, P < 0.01) and α-SC9 fibrils (321 days, P < 0.001) prolonged survival by 42% and 39%, respectively. Furthermore, vaccination with α-SC3 fibrils (316 days, P < 0.001) and HET-s fibrils (316 days, P < 0.01) resulted in a 37% increase in survival.

The amount of brain-associated α-syn aggregates in sick mice was not significantly different regardless of their immunization status, whether vaccinated or not, or the route of challenge, intraperitoneal (Supplementary Fig. 6A) or intragastric (Supplementary Fig. 6B). In conclusion, administration of α-syn fibrils via injections into the peritoneum or the wall of the gastrointestinal tract seeded a progressive synucleinopathy in all challenged mice, regardless of their immune status. Within each group, all mice exhibited signs of neurological disease when comparable amounts of α-syn aggregates were present in the brain.

Adult mice (n = 10–12 per group) at an age of 6–8 weeks were anaesthetized with 2%–3% isoflurane and injected intraperitoneally with 200 µl of 100 µg antigen, composed of either HET-s, α-SC3, α-SC6, α-SC8 or α-SC9 fibrils, diluted 1:1 in alum (Alhydrogel adjuvant 2%, InvivoGen) using a 26-gauge needle. Each animal was given four vaccine injections, each spaced 2 weeks apart. Blood samples of 50 µl were obtained from the tail vein of each mouse immediately before each injection and 2 weeks after the final injection. These samples were diluted 1:1 with 5% sodium citrate. The blood was then centrifuged at 500g for 10 min at 4°C, and the resulting plasma was collected and stored at −80°C.

All studies involving animals were approved by the animal protection committee of the North Rhine-Westphalia State Environment Agency (LANUV). All applicable national and institutional guidelines for the care and use of animals were followed.

Previously, we showed that an engineered quadrivalent vaccine mimicking α-syn fibrils induces an immune response against pathological α-syn fibrils by molecular grafting of conformational epitopes present on the surface of α-syn fibrils onto the inert carrier molecule HET-s(218–289) (PDB IDs: 2RNM), the prion domain of the HET-s protein found in the filamentous fungus Podospora anserina.18 Residues 218–289 in the C-terminus of HET-s are unstructured in solution and the non-prion state and can form biologically functional amyloid fibrils when the prion state is adopted.19 HET-s(218–289) fibrils are assemblies of four β-strands forming two coils of a left-handed β-solenoid.20,21 Based on the known structure of two synthetic α-syn fibrils (PDB IDs: 2N0A and 6H6B), we mutated selected amino acid residues in HET-s(218–289) to design four vaccine candidates α-SC3, α-SC6, α-SC8 and α-SC9 (Fig. 1), all of which form amyloid fibrils.

In contrast to our previous study, here we vaccinated groups of animals with either unmodified HET-s fibrils or with only one of the four HET-s-derived fibrillar vaccine candidates, α-SC3, α-SC6, α-SC8 or α-SC9, to assess the level of protection conferred by each vaccine candidate individually (Fig. 1 and Supplementary Fig. 1). Furthermore, in contrast to our previous study, in which we used 25 µg doses of each vaccine, we used 100 µg doses here, representing a 4-fold increase in the dose administered.

In addition to the increased amount of a single vaccine component, it is also conceivable that removing the other HET-s-based antigens reduced potential immunological interference. In the quadrivalent formulation, immune responses directed towards any of the additional vaccine components might have competed for immune attention, whereas single-component vaccines might have allowed more focused responses towards epitopes shared across several α-syn species, including the proposed serine-rich conformational stretch.

B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice (TgM83+/− mice) hemizygous for a transgene encoding human α-syn with the familial A53T mutation were acquired from The Jackson Laboratory and crossed with wild-type C57BL/6J mice to obtain hemizygous offspring.

Adult mice (n = 10–12 per group) at an age of 6–8 weeks were anaesthetized with 2%–3% isoflurane and injected intraperitoneally with 200 µl of 100 µg antigen, composed of either HET-s, α-SC3, α-SC6, α-SC8 or α-SC9 fibrils, diluted 1:1 in alum (Alhydrogel adjuvant 2%, InvivoGen) using a 26-gauge needle. Each animal was given four vaccine injections, each spaced 2 weeks apart. Blood samples of 50 µl were obtained from the tail vein of each mouse immediately before each injection and 2 weeks after the final injection. These samples were diluted 1:1 with 5% sodium citrate. The blood was then centrifuged at 500g for 10 min at 4°C, and the resulting plasma was collected and stored at −80°C.

Animals (n) | 19 | 11 | 12 | 12 | 12 | 11 | 11

Animals (n) | 19 | 10 | 11 | 11 | 11 | 10 | 11

For intraperitoneal challenge, 50 μg of sonicated α-syn fibrils in 20 μl PBS were injected into the peritoneum of TgM83+/− mice (n = 11–12 per group) anaesthetized with isoflurane, using a 30-gauge disposable hypodermic needle. For gut wall injections of α-syn fibrils in TgM83+/− mice (n = 10–12 per group), an aseptic laparotomy was performed. The mice received a subcutaneous injection of buprenorphine (0.05 mg/kg, Bayer) for analgesia, followed by anaesthesia with isoflurane. The abdomen was shaved, disinfected, and a small incision was made with a scalpel. α-Syn fibrils were injected into the wall of the pylorus and duodenum at four points, spaced 0.5 cm apart, using a 10 µl Hamilton syringe. Each site received an injection of 6.25 µg (2.5 µl) of α-syn fibrils. The abdominal wall was sutured, and the skin was closed with wound clips, which were removed 2 weeks later. After surgery, the animals received a single subcutaneous dose of carprofen (5 mg/kg) and were provided with a mix of metamizole (0.5 mg/ml, WDT) and 10% (w/v) sucrose in their drinking water for 3 days. The animals were monitored daily for general health and three times a week for signs of neurological disease, including reduced grooming, ataxia, tremor, bradykinesia, akinesia, lethargy, circling, tail rigidity, paraparesis, paralysis, kyphosis and other symptoms. Body weight was recorded weekly. For immunohistochemical analysis, diseased mice were euthanized with ketamine/xylazine and underwent transcardial perfusion with PBS followed by 4% formalin (Sigma) in PBS. The brains were dissected and stored in 4% formalin in PBS for later processing. For biochemical analysis, diseased mice were euthanized by cervical dislocation, and the brains were snap-frozen on dry ice and stored at −80°C for later processing.

In this study, we modelled body-first PD by challenging fully vaccinated TgM83+/− mice with injections of synthetic α-syn fibrils either into the peritoneum or into the wall of the stomach and pylorus, which seeds α-syn aggregation in the enteric nervous system first.

For intraperitoneally challenged mice, immunization with all vaccine candidates, with the exception of α-SC8 fibrils (236 days, P = 0.063), significantly prolonged survival in comparison to unvaccinated control mice with a median survival of 223 days (Fig. 2B and Table 1). Survival was most prolonged, by 38%, in mice immunized with α-SC6 fibrils (307 days, P < 0.0001). It is noteworthy that vaccination with unmodified HET-s fibrils (261 days, P < 0.05) also significantly protected mice from disease upon challenge, with a slightly more pronounced effect than that observed with α-SC3 fibrils (249 days, P < 0.05). We observed the most effective protection of vaccination in mice challenged with α-syn fibrils via the intragastric route (Fig. 2C and Table 1). In comparison to unvaccinated mice with a median survival of 231 days, vaccination with α-SC8 fibrils (328.5 days, P < 0.01) and α-SC9 fibrils (321 days, P < 0.001) prolonged survival by 42% and 39%, respectively. Furthermore, vaccination with α-SC3 fibrils (316 days, P < 0.001) and HET-s fibrils (316 days, P < 0.01) resulted in a 37% increase in survival.

Censored animals (n) | 1 | 0 | 1 | 0 | 1 | 0 | 0

For intraperitoneally challenged mice, immunization with all vaccine candidates, with the exception of α-SC8 fibrils (236 days, P = 0.063), significantly prolonged survival in comparison to unvaccinated control mice with a median survival of 223 days (Fig. 2B and Table 1). Survival was most prolonged, by 38%, in mice immunized with α-SC6 fibrils (307 days, P < 0.0001). It is noteworthy that vaccination with unmodified HET-s fibrils (261 days, P < 0.05) also significantly protected mice from disease upon challenge, with a slightly more pronounced effect than that observed with α-SC3 fibrils (249 days, P < 0.05). We observed the most effective protection of vaccination in mice challenged with α-syn fibrils via the intragastric route (Fig. 2C and Table 1). In comparison to unvaccinated mice with a median survival of 231 days, vaccination with α-SC8 fibrils (328.5 days, P < 0.01) and α-SC9 fibrils (321 days, P < 0.001) prolonged survival by 42% and 39%, respectively. Furthermore, vaccination with α-SC3 fibrils (316 days, P < 0.001) and HET-s fibrils (316 days, P < 0.01) resulted in a 37% increase in survival.

Interestingly, not only the four vaccine candidates displaying grafted conformational epitopes present on α-syn fibrils, but also unmodified HET-s fibrils induced significant immunity in both body-first PD models. Amino acid residues S227 in β1a in the first rung of HET-s and S263 in β3a in the second rung of HET-s form a continuous stretch of serine residues (Fig. 1F and Supplementary Fig. S8A, B), hence a conformational epitope across subunits within the HET-s fibril.20 Several such stretches of serine residues that form conformational epitopes across subunits are also present in the α-syn fibrils we generated to challenge TgM83+/− mice (PDB ID: 8OQI), where they are formed by S42 or S87 in α-syn (Supplementary Fig. 8),23 which might explain how antibodies to unmodified HET-s fibrils could induce immunity to α-syn fibrils.

As an indicator of motor performance, we measured the grip strength of immunized and non-immunized TgM83+/− mice at 3 and 7 months after challenge with α-syn fibrils and calculated the relative grip strength (Fig. 3C and D and Table 2). In animals injected intraperitoneally with α-syn fibrils, all immunized animals exhibited a significantly higher relative grip strength (100%–109%) than non-immunized animals (86%). Likewise, in animals injected with α-syn fibrils into the intestinal wall, the relative grip strength of immunized animals (102%–108%) was also significantly greater than that of non-immunized animals (91%). In conclusion, following challenge with α-syn fibrils, immunized animals demonstrated a sustained increase in grip strength throughout the disease process, indicating a vaccine-induced protection and improvement in motor function.

Analysis of the relative lifespan without weight loss (Supplementary Table 3) showed that these values do not scale directly with the survival extensions reported in Table 1. After intraperitoneal challenge, α-SC6 produced the strongest survival benefit (38%) but only a moderate increase in the proportion of life spent without weight loss (72% mean-based; 65% median-based), whereas α-SC9, despite a smaller survival extension (29%), showed the highest relative preservation of weight (80% and 88%). HET-s (17%) and α-SC3 (12%) displayed relative values in the range of 66%–71%, which broadly paralleled their modest survival effects. In contrast, α-SC8, despite providing only a 6% survival extension, still showed moderately high relative weight-stable values (75% and 71%). Similar dissociations were observed after intragastric challenge. Together, these findings show that the timing of weight loss does not necessarily extend in parallel with overall survival and suggest that the constructs might differentially influence presymptomatic versus symptomatic phases rather than uniformly shifting the disease trajectory.

The amount of brain-associated α-syn aggregates in sick mice was not significantly different regardless of their immunization status, whether vaccinated or not, or the route of challenge, intraperitoneal (Supplementary Fig. 6A) or intragastric (Supplementary Fig. 6B). In conclusion, administration of α-syn fibrils via injections into the peritoneum or the wall of the gastrointestinal tract seeded a progressive synucleinopathy in all challenged mice, regardless of their immune status. Within each group, all mice exhibited signs of neurological disease when comparable amounts of α-syn aggregates were present in the brain.

To ascertain whether immunization had induced antibodies to synthetic α-syn fibrils, we used ELISA to measure the presence of antibodies in plasma samples from mice that had been immunized and those that had not (Fig. 4B). We observed that each of the four HET-s-derived vaccine candidates, in addition to HET-s fibrils, induced antibodies that recognized synthetic α-syn fibrils. The immune responses induced by HET-s, α-SC6, α-SC8 and α-SC9 fibrils were comparable to each other. Nevertheless, antibodies induced by α-SC3 fibrils demonstrated a slight superiority in recognizing synthetic α-syn fibrils when compared with those induced by α-SC6 (P < 0.01), α-SC8 (P < 0.01) and α-SC9 fibrils (P < 0.001).

In a competitive ELISA (Supplementary Fig. 7), we tested antibodies in plasma from mice immunized with HET-s (Fig. 4C), α-SC3 (Fig. 4D), α-SC6 (Fig. 4E), α-SC8 (Fig. 4F) and α-SC9 fibrils (Fig. 4G) for their ability to recognize pathological α-syn in brain homogenates from patients with DLB (n = 3), MSA (n = 3) or PD (n = 3) when tested against five brain homogenates from non-neurological (healthy) controls (Supplementary Table 1). It is noteworthy that HET-s and α-SC3 fibrils did not appear to induce antibodies against α-syn aggregates present in the brains of patients with DLB. The α-SC3 fibrils induced a mild (ΔOD450 nm = 2) to intermediate (ΔOD450 nm = 4) antibody response, and the HET-s fibrils induced only a mild response to α-syn aggregates in the brains of patients with MSA and PD. The α-SC6 and α-SC9 fibrils induced a robust (ΔOD450 nm ≥ 4) immune response to α-syn aggregates in the brains of patients with PD, a mild to intermediate response to α-syn aggregates in the brains of patients with MSA, and a mild response to those in the brains of patients with DLB. The α-SC8 fibrils exhibited a mild to intermediate response to α-syn aggregates in the brains of patients with MSA, and a mild response to α-syn aggregates in the brains of patients with DLB and PD.

When we tested antibodies in plasma from immunized mice against brain homogenates from patients with synucleinopathies using competitive ELISA, we observed different results compared with when we tested the antibodies against sarkosyl-insoluble fractions prepared from brain homogenates that are enriched in α-syn fibrils.

The authors L.M., V.P., S.R. and G.T. were aware of the group assignments at all times. All studies involving animals were approved by the animal protection committee of the North Rhine-Westphalia State Environment Agency (LANUV). All applicable national and institutional guidelines for the care and use of animals were followed.

Unvaccinated mice injected with α-syn fibrils from our previous study also served in this study as controls, because all experiments were initiated at approximately the same time.23 To analyse the vaccine-induced antibody response of TgM83+/− mice, we collected plasma once before and every 2 weeks after each vaccine dose was administered (Fig. 2A).

Outliers were identified and removed using the ROUT function with Q set to 2%.

In addition, we cannot presently determine whether the protective effects observed here reflect sustained antibody activity throughout the course of disease or whether declining antibody titres contributed to the waning protection observed at later time points. We did not assess antibody titres at later time points post-challenge, and a reversal of seroconversion remains a plausible explanation that warrants future investigation. It also remains unknown whether administering an additional booster at these later time points could prolong protection or whether disease progression would already be too advanced for meaningful benefit. Addressing these questions will be important for evaluating whether such vaccines are best suited for prophylactic use, before or during very early peripheral α-synuclein fibrillization, or whether they also possess therapeutic potential once disease processes are underway.

It should be noted, however, that this projection assumes that vaccination is effective either prior to the initial formation of peripheral α-syn fibrils or at very early preclinical stages. Our study examines prophylactic vaccination, and whether similar levels of protection can be achieved once peripheral fibrillization has already begun remains to be determined in future work.

In addition, we cannot presently determine whether the protective effects observed here reflect sustained antibody activity throughout the course of disease or whether declining antibody titres contributed to the waning protection observed at later time points. We did not assess antibody titres at later time points post-challenge, and a reversal of seroconversion remains a plausible explanation that warrants future investigation. It also remains unknown whether administering an additional booster at these later time points could prolong protection or whether disease progression would already be too advanced for meaningful benefit. Addressing these questions will be important for evaluating whether such vaccines are best suited for prophylactic use, before or during very early peripheral α-synuclein fibrillization, or whether they also possess therapeutic potential once disease processes are underway.