Recombinant B Cell Epitopes Aβ1-15 Fusion Antigen Vaccines For Alzheimer's Disease

Alzheimer's disease is one of neurodegenerative diseases which are characterized by progressive loss of memory and a general cognitive decline. The neuropathological features of the disease include neurofibrillary tangles, deposition ofβ-amyloid in senile plaques and neuronal loss in affected brain regions. The Amyloid cascade hypothesis suggests that accumulation of excessive Aβ42/Aβ40 in brain is the main cause of Alzheimer's disease. Accordingly, current active and passive vaccination strategies developed for AD were aimed at reduction of level of Aβin the brain.To develop a safe and effective immunotherapy for AD, vaccines will require a delicate balance between providing specific and adequate anti-AβAb responses sufficient for therapeutic benefit while eliminating adverse T-cell-mediated autoimmune responses. The immunogenic B-cell epitopes Aβ1-15 is critical for clearance of Aβplaques, but that will not stimulate anti-AβT cells. We have reconstructed recombinant epitope fusion vaccines composed of six Aβ1-15, which in tandem with a universal synthetic T cell epitope, pan HLA DR-binding peptide (PADRE), or Hc fragment of botulinum neurotoxin serotype A as carrier.These fusion genes were cloned into the prokaryotic expression vector pTIG-Trx; the purified recombinant proteins (6Aβ, 6Aβ-T, 6Aβ-AHc-C, AHc-5Aβ-T) were used to vaccinate Balb/c mice. The Balb/C mice were immunized with recombinant proteins respectively and high anti-Aβ42 antibody titers were induced. Antibody titers of the recombinant sub-unit vaccines which contained a helper T cell epitope and AHc were significantly higher than that of 6Aβ. Immunization of Balb/C mice with recombinant proteins primarily induced a Th2-based humoral immune response. Dot blot showed that antibodies in sera could bind to the oligomer of Aβbetter than the monomer and fibril form. Recent reports have suggested that the toxicity of Aβand other amyloidogenic proteins does not lies in the insoluble fibrils but rather in the soluble oligomeric intermediates. The idea might have an important significance for our recombinant fusion antigen as AD vaccines which induce sera antibodies binded to the oligomer of Aβ.PDAPP mice were immunized with recombinant proteins 6Aβ-T and 6Aβ-T-AHc -C.Results showed a similar immunogenicity as observed in Balb/C. High antibody levels in serum and Th2 immune response were induced in the immunized mice. The reduction latency in the Morris water maze of the treated mouse indicated that recombinant proteins 6Aβ-T and 6Aβ-AHc-C could improve mouse's cognition. Vaccination significantly reduced Aβburden in several areas of brain in immune mice versus control animals. Theses dates were confirmed by quantified analysis of immunohistochemisty results in the brains of experimental and control mice.Subsequently, we have engineered three DNA epitope vaccines that encoding recombinant fusion Aβ15 gene. To test the immunogenicity of these DNA epitope vaccines in vivo, Balb/C mice were immunized with these recombinant plasmids and humoral immune responses were assessed. Impressively, favourable humoral immune response was detected in the vaccinated mice. Antibody assay showed that the highest antibodiy titers in immunized mice could be reached to 1:6400. It demonstrated that our DNA vaccines polarized the immune response towards an anti-inflammatory Th2 type. Similarly, Dot-blot results showed that serum antibodies produced which primarily bound to Aβoligomers may be related to our fusion genes.Molecular adjutants were used to improve the antibody levels of DNA vaccine. It was demonstrated that supplementation of our DNA epitope vaccine with pVAX1-S-GM-CSF enhanced the humoral immunity and did not change the immune response pathway of our DNA vaccine. According to our results, GM-CSF has an important role on increase the DNA vaccine potency and could be used as molecular adjuvant in DNA epitope vaccines for Alzheimer's disease.In summary, high antibody levels in serum and Th2 immune response were induced in the immunized mice with the recombinant subunit vaccines and DNA vaccines. 6Aβ-T and 6Aβ-AHc-C could improve mouse's cognition and significantly reduced Aβburden in several areas of the brain of immune mice versus control animals. Therefore, the recombinant subunit vaccines could be good candidate vaccines for immunetherapy of human Alzheimer's disease. And the DNA vaccines or combination with the molecular adjuvant GM-CSF might be an alterative candidate vaccine for Alzheimer's disease.