Vaccigrade

PLP-α-syn mice expressing human wild-type α-syn under the control of an oligodendroglia-specific PLP promoter to model the typical pathology of MSA [28 (link)], were maintained on a light/dark cycle of 12 h (lights on at 6 am) with free access to food and water at the Animal Facility of the Medical University of Innsbruck. Six month old male and female PLP-α-syn mice, were randomly allocated to four groups and received over a period of 12 weeks a weekly, intraperitoneal injection of either MPLA (50 μg and 100 μg, vac-mpls, MPLAs vaccigrade, Invivogen, San Diego, CA, USA), LPS (3 μg, Sigma-Aldrich, Vienna, Austria) or vehicle. Importantly, the MPLA preparation used here (vac-mpls) is synthetic lipid A from E. coli, serotype R515, i.e. a pure monophosphoryl lipid A compound produced by chemical synthesis. This preparation activates TLR4 but does not activate TLR2 reflecting its high purity (vaccigrade">http://www.invivogen.com/mplas-vaccigrade). All the experiments were done according to the EU and the Austrian legislation and with permission of the Ethics Board at the Federal Ministry of Science and Research, Austria (permission BMWFW-66.011/0122-WF/V/3b/2014). All analyses were done by a researcher who was blinded to the treatment of the animals.

The multi-epitope peptide vaccine HisDTC was previously designed, as detailed in [23 (link)]. The peptides were synthesized by GenScript Biotech (Leiden, The Netherlands) with purity ≥ 95% and stored at −20 °C until encapsulation.
TLRL-2 (Pam3CSK4) and TLRL-3 (Poly(I:C) HMW (VacciGrade™, InvivoGen, Toulouse, France) were used as adjuvants when needed.

Unless specified, 30 μg OVA together with 20 μg CpG OND 1826 and 20 μg poly(I:C) (VacciGrade, all from InvivoGen) were injected subcutaneously into tumour-bearing mice adjacent to the tumour. For vaccination with BMDCs, 1 million SIINFEKL-loaded LPS-matured BMDCs were injected together with 20 μg CpG and 20 μg poly(I:C). The tumour volume was then measured every one or two days using callipers. The human vaccination trial was performed as previously described^{20 (link)}. The times of vaccination were stratified into vaccinations performed before or after 13:00. The patients included received all of their vaccinations before or after this cut-off time.

To activate STING signaling pathway in vivo, 200 nmol/mouse c-di-GMP (VacciGrade) (Invivogen, San Diego, CA, USA) was either injected (i.v.) 18 h before experiment or injected (i.p.) at days 1, 3, and 5 before experiment.

Female CB6F1 mice (Charles River Laboratories, Montreal, QC, Canada) (5 to 7 weeks old) or Hartley guinea pigs (Medimabs, Montreal, QC, Canada) used for immunization were cared for in accordance with Canadian Council on Animal Care guidelines. Experimental methods were reviewed and approved by the University of Alberta Health Sciences Animal Welfare Committee. Recombinant WT or ΔHVR1 gpE1/gpE2 antigens (2 μg [mouse] or 7.5 μg [guinea pig]) were mixed at a 1:1 ratio in a volume with 75 μg alum and 7.5 μg monophosphoryl lipid A (MPLA) (Vaccigrade; InvivoGen, San Diego, CA, USA). Mice were given intramuscular injections (35-μl final injection volume) at days 0, 14, 28, and 56. Guinea pigs received subcutaneous injections (100-μl final injection volume) at days 0, 14, 42, and 90. Prevaccination blood samples were collected at day 0, and postvaccination blood samples (terminal bleeds) were obtained 14 days after the final immunization. Whole-blood samples were centrifuged at 5,000 × g for 15 min, and sera were collected and heat inactivated at 56°C for 30 min. Serum samples were stored in aliquots at −80°C until use.