| Alzheimer's disease(AD)is an irreversible degenerative disease of the central nervous system,and is the most common form of dementia.The main pathological features of AD include amyloid plaques formed by Amyloid?(A?)peptide,neurofibrillary tangles formed by hyperphosphorylated Tau protein and neuronal death.The pathogenesis of AD has not been fully elucidated.According to the amyloid cascade hypothesis,the accumulation of A?42 peptide and subsequent accumulation and deposition of amyloid plaques are the main causes of the neurodegenerative process of AD.Amyloid plaque accumulation may begin before the appearance of abnormal Tau,and increased amyloid plaque accumulation is associated with subsequent increases in Tau.Therefore,immunotherapy targeting A?has been the focus of AD treatment research.The first A?active vaccine to enter human clinical trials,AN-1792,is treated with human A?42as the immunogen.Its phase II clinical study was halted when 6 percent of patients tested developed meningitis.Subsequent studies found that the immunogen A?42 small peptide of AN-1792 vaccine contained T cell recognition sites,and vaccine immunization caused over-activation of T cells targeting A?42 in patients,leading to T cell infiltration and inflammation in the brain.Nevertheless,a long-term follow-up study of AN-1792 confirmed that it played a positive role in clearing amyloid plaques in the brain.Therefore,in order to ensure the safety of the vaccine,the second generation of A?-targeted vaccine mainly uses N-terminal A?short peptide containing B cell epitopes as the immunogen,hoping to induce safe and effective antibody immune response.However,up to now,almost all immunotherapies against A?have ended in clinical failure,mainly because the vaccine has chosen A?short peptide with high safety as the immunogen,which makes its immunogenicity difficult to reach the level of cure,and the antibody is difficult to play a long-term clearance effect.In order to obtain a safe AD active immunization vaccine with high immunogenicity,we used A?1-6 as the immunogen and selected ferritin nanoparticles that could be loaded with exogenous antigen as the vaccine carrier.Ferritin nanoparticles have the properties of homogeneity,stability,temperature and p H tolerance,and are ideal protein vaccine carriers.It has been widely used in the research of influenza and novel coronavirus vaccines due to its characteristic of assisting antigen formation in trimer form.Based on this,four recombinant ferritin AD vaccines containing different copy numbers of A?1-6 were designed and constructed in this paper.Including 1Copy A?1-6-Ferritin,3Copy A?1-6-Ferritin,10Copy A?1-6-Ferritin,20Copy A?1-6-Ferritin.The results showed that the fusion of different copies of A?1-6 recombinant ferritin at the N-terminal could still form a uniform and stable nanoparticular structure,and the antigen could be displayed on the surface of ferritin particles.After immunizing wild mice with the four candidate A?epitope vaccines,all the candidate protein vaccines could induce the production of antibodies against A?42 in vivo.The serum antibody titer of mice induced by 3Copy A?1-6-Ferritin after four times of immunization was the highest with strong persistence,and the antibody still had positive binding after 10~5 times dilution.This was followed by 20Copy A?1-6-ferritin.Most importantly,none of the four candidate A?epitope vaccines induce T cell immune response against A?42.In conclusion,the preparation method of A?epitope vaccine based on ferritin nanoparticle carrier designed in this paper is simple,with high protein purity and stable particle structure.The 3Copy A?1-6-Ferritin recombinant protein vaccine showed strong immunogenicity,good antibody persistence and high safety in normal mice.Next,we will continue to study its therapeutic effect on cognitive decline and brain amyloid deposition in AD model mice. |
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