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. 2014 Dec 3;9(12):e113585.
doi: 10.1371/journal.pone.0113585. eCollection 2014.

Oral administration of a fusion protein between the cholera toxin B subunit and the 42-amino acid isoform of amyloid-β peptide produced in silkworm pupae protects against Alzheimer's disease in mice

Si Li  1 , Zhen Wei  2 , Jian Chen  3 , Yanhong Chen  2 , Zhengbing Lv  3 , Wei Yu  3 , Qiaohong Meng  4 , Yongfeng Jin  5
Affiliations

Oral administration of a fusion protein between the cholera toxin B subunit and the 42-amino acid isoform of amyloid-β peptide produced in silkworm pupae protects against Alzheimer's disease in mice

Si Li et al. PLoS One. .

Abstract

A key molecule in the pathogenesis of Alzheimer's disease (AD) is a 42-amino acid isoform of the amyloid-β peptide (Aβ42), which is the most toxic element of senile plaques. In this study, to develop an edible, safe, low-cost vaccine for AD, a cholera toxin B subunit (CTB)-Aβ42 fusion protein was successfully expressed in silkworm pupae. We tested the silkworm pupae-derived oral vaccination containing CTB-Aβ42 in a transgenic mouse model of AD. Anti-Aβ42 antibodies were induced in these mice, leading to a decreased Aβ deposition in the brain. We also found that the oral administration of the silk worm pupae vaccine improved the memory and cognition of mice, as assessed using a water maze test. These results suggest that the new edible CTB-Aβ42 silkworm pupae-derived vaccine has potential clinical application in the prevention of AD.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The gene and protein sequences of CTB- Aβ42.
The box contains the amino acid GPGP linking CTB and the Aβ42 gene.
Figure 2
Figure 2. Schematic structure(A), restriction enzyme digestion and PCR identification(B) of recombinant plasmid pBacPAK8-CTB-Aβ42.
(A) LB, left border; AcMNPV, Autographa Californica Nuclear Polyhedrosis Virus; Ph-Pro, AcMNPV polyhedrin promoter; L, linker peptide (GPGP); poly A+, polyadenylation signal; RB, right border. (B) M, DNA marker DL 2000 (TakaRa); lane 1, recombinant plasmid digested with BamHI and Xho I; lane 2, recombinant plasmid digested with BamHI; lane 3, recombinant plasmid digested with Xho I; lane 4, PCR amplification of recombinant plasmid.
Figure 3
Figure 3. Quantitative analysis of protein production levels in BmN cells.
Data are presented as the mean concentrations.
Figure 4
Figure 4. Quantitative analysis of protein production levels in silkworm pupae.
Data are presented as the mean concentrations.
Figure 5
Figure 5. Western blot analysis of the CTB-Aβ42 fusion protein expressed silkworm pupae.
(A) Lanes 1: unboiled silkworm pupa sample infected with linearized virus, Lanes 2: unboiled silkworm pupa sample infected with recombinant virus; (B) Lanes 1: boiled silkworm pupa sample infected with linearized virus, Lanes 2: boiled silkworm pupa sample infected with recombinant virus.
Figure 6
Figure 6. Determination of the affinity between the CTB-Aβ42 protein and GM1 by GM1–ELISA.
(A) CTB-Aβ42 fused protein derived from silkworm pupae infected with recombinant virus (circles), positive control, CTB standard protein samples (squares) and negative control, silkworm pupae infected with linearized virus(triangles). (B) The three mentioned samples, both boiled and non-boiled, with the same amounts (50 ng CTB/ml) were measured for A492 signal levels. The results are presented as the means ± SE.
Figure 7
Figure 7. Serum anti-Aβ42 antibody titers.
The results are presented as the mean titer values. * P<0.05, ** P<0.01, versus mice of control group.
Figure 8
Figure 8. Learning curves showing the average latency (mean ± SE) to find the platform (days 1–5).
Figure 9
Figure 9. Annulus crossing index during the probe trial after the last training trial on the 6th day.
* P<0.05, versus mice of control group.
Figure 10
Figure 10. Immunostaining of B6C3-Tg mouse brains.
Left, frontal lobe (Bar  = 100 µm); middle, hippocampus (Bar  = 100 µm); right, cortex (Bar  = 20 µm).
Figure 11
Figure 11. Levels of Aβ42 peptides in the brain.
* P<0.05, versus mice of control group.
See this image and copyright information in PMC

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Grants and funding

This work was financially supported by research grants from the Natural Science Foundation of Zhejiang Province (Y3110354), National High Technology Research and Development Programs of China (2012ZX09102301-009, 2011AA100603), the Open Fund of Zhejiang Provincial Top Key Discipline of Biology, and the Project of Education Department of Zhejiang Province (Z201018783, Y200909617). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.