Study On The Immunotherapeutic Effect Of A Norovirus P Particle-based PTau31 Epitope Vaccine For Alzheimer’s Disease

Alzheimer’s disease(AD)is a progressive neurodegenerative disorder and becomes the most common form of dementia among the elderly in contemporary society.The neuropathological features of AD include extracellular senile plaques formed by amyloid-β(Aβ),intraneuronal neurofibrillary tangles(NFTs)accumulated by hyperphosphorylated Tau protein,and widespread neuronal loss.For a long time,the “amyloid cascade hypothesis” posits that abnormal accumulation of Aβ peptide in the brain is central to the onset and progression of AD.However,none of the Aβ immunotherapies focusing on reducing Aβ deposition have produced clinically meaningful results to date.Hence,it is becoming clear that only elimination of Aβ pathology is insufficient to cure AD,and the development of tau-targeted treatment has become one of very potential therapeutic strategies.Tau protein is a major microtubule-associated protein(encoded by MAPT gene)in the mammalian nervous system that regulates the assembly and stability of microtubules and axonal transport under physiological conditions.However,under pathophysiological conditions,abnormal hyperphosphorylation of tau at numerous toxic epitopes(such as Ser202,Thr205,Ser238 and Ser404)has been extensively reported in the context of AD and related tauopathies,including corticobasal degeneration(CBD),progressive supranuclear palsy(PSP),Pick’s disease(PD),and frontotemporal lobar degeneration(FTLD).The genetic coding defect of MATP and hyperphosphorylatical tau protein induced disease are termed to tauopathies.To date,the main cause of tau hyperphosphorylation in sporadic tauopathies remains uncertain.In familial tauopathy patients,genetic mutations including G272 V,P301L,P301 S,V337M and R406 W have been identified,which predispose hyperphosphorylation of tau protein,thereby compromising the ability of tau to promote microtubule assembly.These mutations promote the aggregation of tau to form paired helical filaments(PHFs)and neurofibrillary tangles(NFTs).To research the pathological progression of tauopathy in vivo and develop drugs based on tauopathy,a serious of animal models which express the mutated human tau gene have been developed by scientists.These tau transgenic mice mouse models have some important attribute of AD pathology,such as synaptic pathology,NFT formation and neuron loss.These transgenic mice have become the essential tools for exploring the mechanism of tau dysfunction and developing the therapeutics for neurodegenerative diseases.Among these,the TauP301S(PS19line)is a wild used AD tauopathy mice model.Human tau protein expression levels in P301 S Tg mice were approximately five-fold higher than that of endogenous mouse tau,leading P301 S Tg mice to develop early and robust manifestations of neurodegenerative tauopathies.Tau filaments develop in P301 S Tg mice at 6 months of age and are progressively enriched in parallel with prominent neuronal death and brain atrophy by 9-12 months of age.However,at the present time,the P301 S transgenic mice are wildly applied in the pre-clinical valuation of drugs for AD,these tests usually use behavioral and pathological experiments to assess the effect of drugs,but less knowledges have been explicit in the changes of behavioral,pathological and serological tendency as mice aging,as well as the relationship among these indicators.On the other hand,the epidemiological studies on dementia discovered that the male and female people have great differences between the morbidity,symptoms and survival in Alzheimer’s disease,however,the sex differences in the reaches of drugs based on P301 S mice model have not been concerned.In the current study,the tau-targetted active immunotherapy is a novel therapeutic strategy of AD.However,the vaccines with tau epitopes under pre-clinical and clinical trails only consist up to two phosphorylated site,which induced limited therapeutic effect.And how can active vaccines keep high-titer and long-termed antibodies in vivo also remains to be explore.In addition,the fuctional mechanism of vaccines had not been deeply discussed.In the current study,we systematically investigated the discrepancies in behavior,tau pathology,and plasma and brain biomarkers between TauP301 S transgenic mice of different sexes and ages and examined the correlations between these factors during tauopathy progression,and we have also screened some potential biomarker which can indicate the progress of tau pathology in P301 S mice.The result shows that the male P301 S transgenic mice perform significant changes in weight loss,survival rate,clasping,kyphosis,composite phenotype assessment,nest building performance,tau phosphorylation at Ser202/Thr205 and astrocyte activation compared to that of wild type littermates.In contrast,female P301 S transgenic mice were only sensitive in the Morris Water Maze and open field test.In addition,we characterized the absence of macrophage-inflammatory protein(MIP-3α)and the upregulation of interferon(IFN)-γ,interleukin(IL)-5,and IL-6 in plasma,these factors can be served as potential AD biomarker.The current development of tau-epitope vaccines in the pre-clinical and clinical phases are focus on up to two phosphorylated tau site,inducing a limited cure effect.In addition,to elicit a high level and long-termed antibodies in vivo,high-efficient vaccine carrier and suitable adjuvant of vaccine remain to be discussed.to solve these problems,we developed a norovirus P particle-based phosphorylated tau-targeted active vaccine for AD therapy,and analyzed its immunogenicity,safety and efficacy in P301 S mice.To screen the optimal epitopes of tau vaccine,we combined the truncated peptides around highly phosphorylated tau sites in AD patients,and screened a synthetic pTau31 peptide which owns phosphorylated tau epitopes on Ser202,Thr205,Ser396 and Ser404.This peptide can induce all these four epitopes-specific antibodies,with no T cell immunoreaction targeting phosphorylated and non-phosphorylated tau.Then we designed the method to connect the pTau31 peptides to the norovirus P particle protein by cysteine/ air oxidation successfully,and named it as PP-pTau31.We chose the combination of CpG and AS02 as the optimal adjuvant among several candidate adjuvants in for the usage of PP-pTau31 active vaccine,which can elicit high-level and long-termed antibodies in vivo with no T cell immunoreaction risks.We also explored the immunogenicity,effectiveness and safety of PP-pTau31 vaccine in the P301 S transgenic mice.The result shows that the PP-pTau31 vaccine can successfully decrease the accumulation of pTau in brain,decline the gliosis in brain,alleviate tauopathy-relevant dyskinesia and cognitive impairment,and did not induce the T cell immunoreaction to phosphorylated and non-phosphorylated tau,as well as no abnormal changes of inflammatory cytokines,chemokines and organs.Last,we also explore the the mechanism of action of antibodies induced by the PP-pTau31 vaccine.The results show that the pTau31-specific antibodies may act their functions via multi-pathway:(1)the antibodies in blood bind to free pTau in blood,and induce a concentration gradient of pTau between blood and brain,which promote the pTau in brain efflux into blood,and decline the load of pTau in brain;(2)part of antibodies in blood cross the blood-brain barrier,and bind to the free pathological tau in brain to inhibit its toxicity to neurons;(3)part of antibodies in blood cross the blood-brain barrier,bind to NFT in brain and from antigen-antibody conmplex,following by being degraded and declining the tauopathy.Summary,we successfully completed the behavioral,pathological and serology study on the TauP301 S transgenic mice,mastered the relationships between these indictors and disease progression,and discovered the some biomarkers such as MIP-3α.On the bases of these,we further completed the development of a kind of norovirus P particle-based pTau31 epitope vaccine,PP-pTau31.The results shows that this vaccine can induce high-level and long-termed pTau-specific antibodies in vivo,and decline the characters on behavior and pathology,with no risks of T cell immunoreaction,abnormal levels of cytokine and visceral injury.