Establishment And Immunogenicity Evaluation Of Norovirus P Particles-based Chimeric Alzheimer’s Disease Protein Vaccine

Alzheimer’s disease(AD) is a progressive age-related neurodegenerative disorder. The most common early symptom is difficulty in remembering recent events. As the disease advances, symptoms can include problems with language, disorientation, not managing self-care and behavioural issues. Gradually, bodily functions are lost, ultimately leading to death. By the year 2015, AD has affected more than 46.8 million AD patients worldwide, which has become the most important health issue for the elderly. Besides, it also brings heavy mental stress and medical care burden to the patient family and the society. However, till now, Alzheimer’s disease cannot be cured or delayed, which results in the serious damage to the old people’s health and life quality.Alzheimer’s disease has three major pathological hallmarks, including the extracellular amyloid plaques deposition in the brain cortex and hippocampus, intracellular neurofibrillary tangles formed by hyperphosphorylated tau and neuron loss. At present, the etiology and pathogenesis of AD has not been clarified, the majority of researchers consider that the excessive deposition of Aβ42 protein in brain plays a key role in the development of Alzheimer disease(AD). Thus, active Aβ immunotherapy which has potential for preventing cerebral amyloid plaques has emerged as the main therapy for AD. However,one clinical trial of vaccination with synthetic human Aβ1-42(AN1792) in AD patiens resulted in the development of meningoencephalitis in 6% patients and the low titer of anti-Aβ42 antibody. Since T cell epitopes of Aβ peptide reside in amino acid 16-30, AD patients immunized with Aβ1-42 vaccine had strong Aβ-reactive T cell responses and finally developed self-limited aseptic meningoencephalitis. Compared with Aβ1-42, Aβ1-6 polypeptide contains only B cell epitope but no T cell epitope. Several preclinical studies demonstrated that the Aβ epitope vaccine could elicit similar therapeutic effects as Aβ1-42 vaccine without T-cell activation, so Aβ1-6 epitope vaccine is a promising AD vaccine.The vaccine vector is also very important for the immunization effect, an ideal platform can efficiently enhance the immunogenicity of the vaccine. The P domain protein of Norovirus capsid is a novel vaccine vector. It could form dimer, 12-mer, 24-mer and 36-mer protein in vitro. The 24-mer protein structure is the main form of P domain protein and is named of P particle. P domain protein has three protruding loop structures(loop1, loop2, loop3). The structural analysis of the P domain protein revealed that those three surface loops were on the distal surface of each P domain, which are located on the outermost surface of the P particle. P particle could induce high immunogenicity and is extremely stable, and it could be expressed in E. coli cells, which make it an efficacious vaccine platform. Most importantly, the protruding surface loops of P protein are suitable for the foreign antigens insertion, so the P particle could fully present antigens on its surface and greatly increase the immunogenicity. Taken together, Nov P particle is a very ideal antigen display platform.In this study, we used the P domain protein of NoV Hunter strain as the template, created a new restriction enzyme cut site(EagI) on the P domain by site-directed mutagenesis without changing the original sequence of the P domain. The synthesized DNA fragment 1copy-Aβ1-6-loop2 or 10copy-Aβ-1-6-loop2 was cloned into the plasmid pET28a-P domain, resulting in the recombinant plasmids pET28a-P particle-1copy-Aβ1-6-loop2 and pET28a-P particle-10 copy-Aβ1-6-loop2. After that, we expressed those two P particle-based constructs in E. coli strain BL21 and the recombinant protein was purified by Ni-NTA affinity and Superdex 200 gel filtration chromatography. The purified recombinant PP proteins were also analyzed by to detect its MW and by electron microscopy to confirm its aggregation state. Furthermore, we checked the immunogenicity and safety of those two AD protein vaccine candidate in C57BL/6J mice. We found that both two AD protein vaccine could elicit high titers of Aβ42 specific antibody. In addition, the P particle based AD protein vaccine could not activate Aβ42 specific T-cell response, indicating that those two AD protein vaccines have the strong immunogenicity and high safety. Taken together, we successfully constructed, expressed and purified two kinds of P particle based AD protein vaccines. They could induce high Aβ42 titers without T-cell activation. Next, we will check the efficiency of those AD protein vaccines in APP transgenic mouse.