Establishment And Immunogenicity Evaluation Of Bacterium-like Particle-based Chimeric Alzheimer's Disease Protein Vaccine

Alzheimer's disease(AD),the most common cause of dementia in the elderly,is a neurodegenerative disorder neuropathologically characterized by extracellular aggregation of amyloid-?(A?)plaques,intracellular aggregation of hyperphosphorylated tau protein causing neurofibrillary tangles,and extensive neuronal loss.It is hypothesized that the overproduction and accumulation of A? is closely related to the onset and progression of AD.According to the amyloid-cascade hypothesis,A? is the trigger of AD,and tau pathology and other degenerative changes are the downstream consequence of A? pathology.As the main component of senile plaques,A? is produced by sequential cleavage of the amyloid precursor protein(APP)and exists in different aggregated forms,among which the soluble forms are thought to be the most toxic forms responsible for the impairment of synaptic and cognitive function.Therefore,reducing the level or blocking the toxicity of A? in the brain is a potential therapeutic strategy.Over the past decade,many novel therapeutic strategies have been utilized,among which active and passive immunization were demonstrated to be the most advanced approaches.Many active immunizations against A? and passive transfer of monoclonal A? antibodies have been performed in various AD mouse models and indicated an effective reduction in the cerebral A? load and improvement in cognitive impairment.However,the first Phase IIa clinical trial for an active immunization vaccine(AN1792),which used the full length synthetic human A?1-42 peptide as an immunogen,was halted due to the development of meningoencephalitis in 6% of the vaccinated patients.This was thought to be caused by the A?-specific T-cell autoimmune response,and the T-cell epitopes were later demonstrated to be located in the C-terminal(A?15–42)of A?1-42.Therefore,subsequent A? immunotherapies used short or fragmented A? peptides in the N-terminal of A?1-42 as an immunogen in order to avoid the activation of A?-specific T cells.For example,CAD106 used a small N-terminal A?1-6 amino acid peptide fragment as a B-cell epitope,thereby inducing efficacious A? antibodies titers in animals without causing a T-cell response,and,importantly,the vaccine reduced amyloid accumulation in an APP transgenic mouse model.In regard of safety and efficacy,we selected the A?1-6 amino acid peptide fragment as the epitope in our study.An important goal of active immunization is to induce high levels of antibodies in vivo,and active immunotherapy against AD is no exception.High levels of A?-specific antibodies are conducive for the clearance of A? plaques and reduction of the A? load in the brain.As a hapten,A?1-6 is not immunogenic in itself,and requires coupling with an ideal carrier to induce high levels of antibodies.Bacterium-like particles(BLPs),previously referred to as gram-positive enhancer matrix particles,are non-living and have the same shape and size as the living bacterium but consist of peptidoglycan shell particles instead of the intracellular content of Lactococcus lactis bacteria,and have achieved the Generally Recognized As Safe(GRAS)status from the Food and Drug Administration(FDA).BLPs can serve as a multifunctional and safe carrier system,with a high loading capacity for subunit protein vaccines,and antigens can be specifically loaded on the surface of BLPs via fusion with a peptidoglycan anchoring domain(PA).The PA is derived from the C-terminal of the lactococcal cell-wall hydrolase AcmA,and can be bound to the peptidoglycan surface of BLPs via non-covalent binding with high affinity.BLPs have been studied for vaccination against the malaria parasite antigen,the pneumococcal antigen,swine type O,the foot-and-mouth disease virus antigen,and the influenza antigen,and these studies have demonstrated that BLPs induced a higher immune response than did the antigen alone.To enhance the immunogenicity of A?1-6,we fused one,three,six,or nine copy numbers of A?1-6 to the PA.The four recombinant proteins were successfully expressed by Escherichia coli and bound to the surface of the BLPs,and four BLP-based A? vaccines were obtained.All of the four vaccines induced high levels of A?-specific antibodies in mice.Most importantly,none of these vaccines induced a T-cell-mediated immune response.Among these vaccines,the 6copy-A?1-6-PA-BLP was the most effective in inducing A?-specific antibodies.In addition,the antibodies induced by all the vaccines blocked the toxicity of A?42 oligomers in cell culture.Furthermore,A?-specific antibodies induced by 6copy-A?1-6-PA-BLP were still detected 3 months after the last administration.In view of the safety and the high efficiency in inducing A?-specific antibodies,the BLPs based A?1-6 vaccines may be potential vaccines against AD.