Oral Administration Of Aβ42,CTB-Aβ42 And CTB-Aβ15 Produced In Silkworm Pupae Protects Against Alzheimer’s Disease In Mice

According to the "amyloid cascade hypothesis",P Amyloid protein(AP) deposition, the formation of senile plaques in the brain and amyloid angiopathy is considered to be the key links of the pathogenesis of AD. Senile plaques is mainly composed of Aβ40 and Aβ42 peptide, A(342 peptide is one of the most important toxic ingredients. In order to adopt active Aβ immunotherapy to curb the brain Aβ42 sedimentary formation or speed up the process of eliminating it, which is beneficial to achieve the purpose of prevention and treatment of AD, this study attempts to express Aβ42, CTB-Aβ42 and CTB-AP15 in silkworm pupae by silkworm bio reactor and tests the silkworm pupae-derived oral vaccination containing Aβ42, CTB-Aβ42 and CTB-Aβ15 respectively in a transgenic mouse model of AD. Water maze, immunohistochemical, Elisa, image analysis technology were used to study the prevention and treatment of AD by the silkworm pupae-derived oral vaccination containing Aβ42, CTB-Aβ42 and CTB-AP15 from the behavior, cellular and molecular level.Aβ42, CTB-Aβ42 and CTB-Aβ15 were successfully expressed in silkworm cell ,5th instar larva and pupae respectively by using silkworm bioreactor. The expression level of Aβ42 protein can reach to 2.2 pg/2 x 106 cells in BmN cells,2 μg/mL in haemolymph of 5th instar larva and 0.3 μg/g in silkworm pupae; The expression level of CTB-Aβ42 protein can reach to 10 μg/2 x 106cells in BmN cells,8 μg/mL in haemolymph of 5th instar larva and 0.5μg/g in silkworm pupae; The expression level of CTB-Aβ15 protein can reach to 5 μg/2 x 106 cells in BmN cells,0.8 μg/mL in haemolymph of 5th instar larva and 10 μg/g in silkworm pupae.Silkworm pupae has advantages of easy operation, convenient recycling and high expression level, so as a "bioreactor" for the production of Aβ proteins, the silkworm is a good choice.Anti-Aβ42 antibodies were induced in the B6C3-Tg mice by taking silkworm pupae-derived vaccination containing Aβ42, CTB-Aβ42 and CTB-Aβ15 orally which have significant difference with control group that only take the physiological saline (CTB-Aβ42 group:1:420, CTB-Aβ 15 group:1:390, Aβ42 group:1:300, Control group:1:100).Anti-Aβ42 antibodies titers induced by CTB-Aβ42 and CTB-Aβ 15 pupae-derived vaccination were higher than silkworm pupae-derived vaccination containing Aβ42 only. In water maze probe trial test, the performance of the three vaccinated groups significantly improved over the 5 training days. The escape latency was significantly shorter for the CTB-Aβ42-vaccinated mice and CTB-Aβ 15-vaccinated mice as opposed to the control group on the fourth and fifth days. On the sixth day, The CTB-Aβ42-vaccinated group and CTB-Aβ 15-vaccinated group and the Aβ42-vaccinated group spent more time in the target quadrant (TQ) than did the control group after the platform was removed. CTB-Aβ42-vaccinated group crossed the former location of the platform significantly more often than did the control group (CTB-Aβ42 group:2.2, CTB-Ap 15 group:1.8, Aβ42 group:1.8, Control group:0.6). Compared with the control group mice, Aβ, CTB-Aβ42 and CTB-Aβ 15-fed mice displayed a reduced amyloid-β content in the frontal lobe, hippocampus and cortex in immunohistochemical analysis of senile plaques in the brain. We observed that brain tissues from the mice of the control group or the Aβ42 group developed large amyloid plaques in the frontal lobe, hippocampus and cortex. However, brain tissues from the mice of the CTB-Aβ42 and CTB-Aβ15 group showed small and isolated amyloid plaques in the frontal lobe, hippocampus and cortex. Quantitative immunohistochemistry showed a significant decrease in the percent area of immunoreactivity to Aβ antibodies in the brain of the three vaccinated groups compared to control group mice (Aβ42-fed group p< 0.05, CTB-Aβ42-fed group and CTB-Aβ15-fed group p< 0.01). The Aβ42 level (from both the soluble and Tris-insoluble fractions in the brain) was significantly lower in the CTB-Aβ42-fed group and CTB-Aβ42-fed group than in the control group.These results suggest that the new edible silkworm pupae-derived vaccine containing Aβ42, CTB-Aβ42 and CTB-Aβ15 has potential clinical application in the prevention of AD.