Immunogenicity of MultiTEP platform technology-based Tau vaccine in non-human primates

Abstract

Pathological forms of Tau protein are directly associated with neurodegeneration and correlate with Alzheimer's Disease (AD) symptoms, progression, and severity. Previously, using various mouse models of Tauopathies and AD, we have demonstrated the immunogenicity and efficacy of the MultiTEP-based adjuvanted vaccine targeting the phosphatase activating domain (PAD) of Tau, AV-1980R/A. Here, we analyzed its immunogenicity in non-human primates (NHP), the closest phylogenic relatives to humans with a similar immune system, to initiate the transition of this vaccine into clinical trials. We have demonstrated that AV-1980R/A is highly immunogenic in these NHPs, activating a broad but unique to each monkey repertoire of MultiTEP-specific T helper (Th) cells that, in turn, activate B cells specific to PAD. The resulting anti-PAD IgG antibodies recognize pathological Tau tangles and Tau-positive neuritis in AD case brain sections with no staining in control non-AD cases. These published data and efficacy results support the AV-1980R/A vaccine progression to first-in-human clinical trials.

Fig. 1. Characterization of the bulk AV-1980R product during manufacturing.

a size exclusion chromatography (SEC) analysis of the AV-1980R protein in 8 M urea in the mobile phase. b Analytical ultra-centrifugation (AUC) profiles of the protein in 6 M urea. c AUC profiles of the protein in 3.2 M urea. d SEC analysis of the protein with no urea in the mobile phase.

Fig. 2. Characterization of purified AV-1980R protein by RP-UPLC, SDS-PAGE assay, and DLS profile.

a Representative chromatogram from reverse-phase ultra-performance liquid chromatography (RP-UPLC): C4 2.1 x 50 mm, 1.7 μm column; Mobile Phase A: 0.1% TFA in water; Mobile Phase B: 0.1% TFA in acetonitrile; Flow rate: 0.5 mL/min, Gradient 0–100%B over 37 min. b SDS-PAGE profiles of the AV-1980R protein in reduced and non-reduced conditions. The main band in all samples corresponds to the expected molecular weight of AV-1980R of 31.6 kDa. The numbers on the left side of the image correspond to the molecular weight of the standard in kilodaltons. c DLS profile of AV-1980R, distribution by mass and intensity.

Fig. 3. AV-1980R/A induced strong cellular responses specific to MultiTEP Th epitopes but not Tau_2-18 peptide in adult non-human primates.

INFγ producing T cells were detected in PBMC of vaccinated NHPs by ELISPOT assay. The individual monkeys are plotted on the x-axis, and the number of INFγ-positive T cells is plotted on the y-axis. The PBMC restimulation is done by either the cocktail of peptides derived from the MultiTEP platform (black bars) or by the Tau_2-18 peptide (white bars).

Fig. 4. AV-1980R/A activated a broad repertoire of Th cells specific to individual Th epitopes comprising MultiTEP in NHPs.

Data presented as the number of IFNγ cytokine-producing cells (SFCs) specific to individual peptides minus the background level. All peptides were used at 20 μg/ml.

Fig. 5. AV-1980R/A induced high titers of anti-Tau antibodies in Macaca fascicularis.

a Endpoint titers of the generated antibodies were evaluated in the sera of individual animals after three immunizations. Horizontal lines indicate the median titer and the x-axis grouping indicates the target-specificities of the generated responses. Titers of generated antibodies specific to MultiTEP are significantly lower than titers of antibodies specific to Tau2-18 (*p = 0.0173, Ordinary One-way Anova) (b) AV-1980R/A induced IgG isotypes of antibodies. Isotypes were analyzed in sera of individual animals diluted 1:2000 after the third immunization. c Dynamics of humoral immune responses in individual animals (n = 6 at the start, #CH2C was euthanized on week 12, and #CH2D was euthanized on week 24) immunized with AV-1980R/A on weeks 0, 2, 6, 26, and 46.

Fig. 6. Characterization of antibodies induced by vaccination with AV-1980R/A in NHP.

a Epitope mapping of immune sera from vaccinated Macaca fascicularis was performed by alanine scanning competition ELISA. Two overlapped epitopes have been detected comprising amino acids 3–9 EPRQEFE and amino acids 7–14 EFEVMEDH. IC₅₀ and the percent of inhibition of antibody binding to Tau_2–18 peptide with mutated peptides (alanine substitution of each single amino acid) is shown in the table. b Relative avidity for binding with Tau protein in mice, rabbits, and monkeys was determined by Sodium thiocyanate displacement ELISA using purified antibodies from pooled sera of individual animals. The effective concentration of NaSCN required to release 50% of primary antibodies from the ELISA plate (half-maximal effective dose (ED₅₀) was 0.8 M for mice, 0.9 M for rabbits, and 1 M for NHP. The concentration of antibodies was adjusted to 30 ng/ml. c Immune sera, but not pre-bleed sera bound to neurofibrillary tangles (NFT) in brain sections from the AD case. Sera were diluted at 1:1000. The original magnification is 40X, and the scale bar is 20 μm. Arrows show NFTs.