Prophylactic Vaccine Based on Pyroglutamate-3 Amyloid β Generates

Nov 22, 2016 - Clearance of amyloid β (Aβ) by immunotherapy is one of the fancy methods to treat Alzheimer's disease (AD). However, the failure of s...
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Letter pubs.acs.org/chemneuro

Prophylactic Vaccine Based on Pyroglutamate‑3 Amyloid β Generates Strong Antibody Response and Rescues Cognitive Decline in Alzheimer’s Disease Model Mice Gao Li,† Zhi-Wen Hu,† Pu-Guang Chen,† Zhan-Yi Sun,† Yong-Xiang Chen,† Yu-Fen Zhao,† and Yan-Mei Li*,†,‡ †

Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China ‡ Beijing Institute for Brain Disorders, Beijing 100069, China S Supporting Information *

ABSTRACT: Clearance of amyloid β (Aβ) by immunotherapy is one of the fancy methods to treat Alzheimer’s disease (AD). However, the failure of some clinical trials suggested that there may be something ignored in the past development of immunotherapy. Pyroglutamate-3 Aβ (AβpE3‑X), which was found to be abundant in the patients’ brain, has attracted much attention after the report that AβpE3‑42 could serve as a template to exacerbate the aggregation of Aβ. In addition, AβpE3‑X could not be recognized by the antibodies targeting the N-terminus of Aβ, suggesting that AβpE3‑X maybe the ignored one. Indeed, passive immunization targeting AβpE3‑X has shown some beneficial results, while active immunotherapy has not been extensively studied. In the present study, we designed and synthesized a novel peptide vaccine targeting AβpE3‑X, which contains AβpE3‑15 as B cell epitope and P2 as T cell epitope. We showed that this vaccine could induce strong antibody response to AβpE3‑X. We also showed that prophylactic immunization of AD model mice with our vaccine could reduce Aβ plaques and rescue cognitive decline. This new kind of Aβ vaccine will open up new directions for AD immunotherapy. KEYWORDS: Alzheimer’s disease (AD), pyroglutamate-3 Aβ, vaccine, prophylactic immunization, Aβ plaques

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assume that clearance of AβpE3‑X may provide benefits for AD patients. Indeed, some researchers have developed different monoclonal antibodies targeting AβpE3‑X, including 9D5,6 mE8,7 and mAb07/1.8 They found that administration of these antibodies to different AD model mice could reduce plaques and rescue cognitive deficits.6−8 Considering the expense of monoclonal antibodies, it would be better to develop an easily available vaccine targeting AβpE3‑X. Gevorkian and co-workers did a pioneering work in this field.9 They used AβpE3‑42 as antigen and found that it could generate strong antibody response specialized to AβpE3‑42 in wild-type rabbits. Taking the failure of AN1792 into account,10

ctive and passive immunotherapy toward Aβ has been extensively studied and is one of the most promising methods to prevent or cure Alzheimer’s disease (AD).1 However, most of the clinical trials targeting Aβ have failed to mitigate the disease progression of moderate AD patients.2 One reasonable speculation is that it is too late to cure AD after irreversible damage to the neuron has been caused by Aβ, suggesting that it might be better to provide prophylactic treatment.2b Another possible reason could be the complexity of Aβ species in the brain.3 It was reported that there were abundant N-terminally truncated or modified Aβ species in the patients’ brain, including pyroglutamate-3 Aβ (AβpE3‑X), which is highly toxic and may act as a template to induce Aβ to form toxic oligomers.4 Recently, it was reported that Bapineuzumab, an antibody targeting Aβ1−5 (DAEFR) that failed in phase III trial,2 could not recognize AβpE3‑X.5 So it is reasonable to © XXXX American Chemical Society

Received: October 3, 2016 Accepted: November 22, 2016 Published: November 22, 2016 A

DOI: 10.1021/acschemneuro.6b00336 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience which was the first clinical trial that used Aβ1−42 as antigen, it should be better to avoid the T cell epitope of Aβ (Aβ16−42)11 in developing vaccines targeting Aβ species. Lemere and coworkers reported their latest work about vaccine targeting AβpE3‑X in their recently published review article and the 2010 annual meeting of Society for Neuroscience.2a They stated that they used AβpE3‑9 linked to KLH as antigen and found some beneficial results. However, carrier proteins like KLH contain too many peptide epitopes and may induce some unnecessary or even bad immune responses.12 In this paper, we report for the first time the development of a peptide vaccine targeting AβpE3‑X based on systematic screening of T cell epitope and B cell epitope. And we demonstrate for the first time that this vaccine can induce strong antibody response and relieve cognitive decline in AD model mice. These results suggest that clearance of AβpE3‑X species based on active immunotherapy could be an effective method for AD treatment.

Figure 1. ELISA of the mixed serum from immunized Balb/c mice.

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RESULTS Selection of T Cell Epitope. To avoid the possible toxic anti-Aβ T cell response (Aβ16−42),10,11 we decided to use AβpE3‑15 as B cell epitope in our first trial. And we used alum adjuvant (Alhydrogel 2%) to facilitate a Th2-biased immune response.13 In order to generate robust immune response, we linked AβpE3‑15 to different T cell epitope peptides or carrier protein. BSA was chosen as a carrier protein and P2 (TT830−843: QYIKANSKFIGITE) and P30 (TT947−967: FNNFTVSFWLRVPKVSASHLE) were chosen as T cell epitopes based on our previous work.12,14 Agadjanyan and co-workers have also shown that Aβ1−12 linked with P2 and P30 could generate robust immune response in Tg2576 mice, guinea pigs and Cynomolgus monkeys.15 So we synthesized AβpE3‑15−P2, AβpE3‑15−P30, and AβpE3‑15−BSA (Scheme 1) and mixed them with Alhydrogel 2% to get the vaccines.

of AβpE3‑15−BSA, we decided to use P2 in our following experiments. Selection of B Cell Epitope and Characterization of the Antibodies. At the beginning, we wanted to develop a vaccine targeting AβpE3‑X specifically. After the selection of T cell epitope, we synthesized AβpE3‑7−P2, AβpE3‑9−P2, AβpE3‑11− P2, AβpE3‑13−P2, and Aβ1−15−P2 (Scheme 1) in order to get one vaccine that can induce antibodies specialized to AβpE3‑X. Balb/c mice were used as described above. The antibody titers against AβpE3‑15, Aβ1−15, AβpE3‑40, and Aβ1−40 were determined (Figure 2). AβpE3‑7−P2 induced similar levels of anti-AβpE3‑15 antibodies compared to AβpE3‑15−P2, while the antibodies induced by AβpE3‑7−P2 showed less cross-reactivity to Aβ1−15 (Figure 2A). It seems that AβpE3‑7−P2 was the vaccine that we wanted. However, when the plates were coated with AβpE3‑40, AβpE3‑15− P2 induced a very high antibody titer against AβpE3‑40, which reached above 400 000, while the titer of AβpE3‑7−P2 was about 8000 (Figure 2B). The high titer of anti-AβpE3‑40 antibodies induced by AβpE3‑15−P2 encouraged us to test this vaccine in AD mouse model. Except for antibodies targeting the N terminus of AβpE3‑X, the serum from mice immunized with AβpE3‑15−P2 may contain antibodies targeting the mid region of Aβ as confirmed by the fact that the antibodies induced by AβpE3‑15−P2 could also bind to Aβ1−40 (Figure 2B). We also analyzed the isotype of the antibodies induced by AβpE3‑15−P2 (Figure 2C). The ratio of IgG1/IgG2a suggested a Th2-biased immune response. Prophylactic Immunization with AβpE3‑15−P2 Significantly Slowed Cognitive Decline in AD Model Mice. Six month old AD model mice8 were immunized for 5 times biweekly with PBS, AβpE3‑15−P2, and Aβ1−15−P2. We wanted to compare our vaccine with the Aβ1−X based vaccine. Wild-type C57BL/6 mice were used as the control group. Each group contained 5 mice. Morris water maze (MWM) test was conducted when the mice reached 12 months old, the results are shown in Figure 3. Compared to AD model mice group, wild-type group and AβpE3‑15−P2 immunized group showed significantly better performance in the training days and testing day (Figure S1 shows more results on testing day). It should be noted that the Aβ1−15−P2 immunized group showed no significant difference

Scheme 1. Peptides and Protein Used in This Study

Balb/c mice were immunized with PBS, AβpE3‑15−P2, AβpE3‑15−P30, and AβpE3‑15−BSA (with adjuvant). Each group contained 4 mice. The antibody titers against AβpE3‑15 were determined by ELISA (Figure 1) 1 week after the final immunization. AβpE3‑15−BSA induced the strongest immune response, while AβpE3‑15−P30 could barely induce any antibody against AβpE3‑15. AβpE3‑15−P2 induced a similar level of antibody titer compared to AβpE3‑15−BSA. Considering the heterogeneity B

DOI: 10.1021/acschemneuro.6b00336 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 3. (A) Escape latency of the mice on training days; *p < 0.05, **p < 0.01, and ***p < 0.001 (# for comparison between AD model mice and WT mice; * for comparison between AD model mice and AβpE3‑15−P2 immunized mice). The error bars for WT group and Aβ1−15−P2 immunized group were omitted to make it easier to read this figure. (B) The number of platform crossings of the mice on day 8. Student’s t test was used to calculate p value.

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Figure 2. (A) Antibody titers determined by ELISA (coated with AβpE3‑15 or Aβ1−15). (B) Antibody titers determined by ELISA (coated with AβpE3‑40 or Aβ1−40). On the horizontal axis, “3-7” refers to the serum from AβpE3‑7−P2 immunized mice, and the rest could be deduced in the same way. (C) Isotype analysis of the serum from AβpE3‑15−P2 immunized group, the dilution used here was 1/51200.

DISCUSSION Passive immunization targeting pyroglutamate-3 Aβ has been shown to be effective in APPswe/PS1ΔE9 mice,8 PDAPP mice,7 and 5XFAD mice.6 These AD model mice have been proven to contain abundant pyroglutamate-3 Aβ plaques especially when they are old.18 Monoclonal antibodies that can specially bind to pyroglutamate-3 Aβ species and cannot recognize unmodified Aβ species were used in these passive immunization studies. It was shown that not only pyroglutamate-3 Aβ species but also total Aβ burdens were decreased after the administration of these monoclonal antibodies. These results highlight the role of pyroglutamate-3 Aβ as a template to facilitate the deposition of Aβ species.2a,4 Active immunotherapy targeting pyroglutamate-3 Aβ has not been extensively studied. Here we described our work in design and optimization of a novel vaccine targeting pyroglutamate-3 Aβ. Alum adjuvant was chosen to induce a Th2 type immune response,13 and the possible toxic T cell response9,10 was avoided by using AβpE3‑15 as B cell epitope instead of the fulllength AβpE3‑X. After experiments on Balb/c mice, we found that AβpE3‑15−P2 induced the highest antibody titer (above 400 000) against AβpE3‑40. This outstanding titer encouraged us to evaluate the effect of AβpE3‑15−P2 on AD model mice. AD model mice immunized with AβpE3‑15−P2 prophylactically

from the AD model mice group. Swim speed of the 4 groups showed no significant difference (Figure S2). Prophylactic Immunization with AβpE3‑15−P2 Significantly Reduced Aβ Plaques in AD Model Mice. After MWM test, the mice were sacrificed, and their brains were sectioned. The sections were stained with 6E10, and the CA3 region was photographed for comparison. The region CA3 of hippocampus is highly associated with memory and cognitive deficits.16 It has been shown that Aβ would disrupt the function of neurons in CA3.17 The results are shown in Figure 4. Compared to AD model mice group, both of the immunized groups showed significantly less Aβ plaques in the CA3 region. And the AβpE3‑15−P2 immunized group showed more reduction compared to the Aβ1−15−P2 immunized group. We have also photographed region CA1 (Figure S3). The results showed similar tendency to those for region CA3. C

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ACS Chemical Neuroscience

with BSA. MALDI-TOF mass showed that one BSA was kinked with about 5 copies of AβpE3‑15 (detailed in Supporting Information). The purity of all the peptides is higher than 95% as confirmed by LC-MS. Peptides for vaccine and BSA−AβpE3‑15 were dissolved in phosphate buffered saline (PBS, pH = 7.4) to 1 mg/mL. Then 100 μL of the solution was mixed with 100 μL of Alhydrogel 2%, which is a kind of Alum adjuvant (Alpha Diagnostic International). Each mouse was intraperitoneally immunized with 200 μL of the final mixture at a time. Control mice were immunized with the same amount of PBS and Alhydrogel 2%. Animals and Immunization. All animal experiments were done with approval and guidance in Laboratory Animal Research Center, Tsinghua University, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). For the selection of T and B epitope, 6−8 week old Balb/c mice were used. Balb/c mice were intraperitoneally immunized with different vaccines 5 times biweekly. One week after the final immunization, mice were sacrificed, and their blood was collected from tail vein. For the prophylactic immunization of transgenic mice, we used double transgenic AD model mice (Mo/HuAPP695 and PSEN1dE9, C57BL/6 background)19 and C57BL/6 mice were used as control. It was reported that this kind of AD model mice would develop abundant Aβ deposits and show impaired spatial learning when they reached 12 months old.20 Immunizations were started when the mice were 6 months old,8 and they were immunized 5 times biweekly. No further treatment was applied to the mice until they grew to 12 months old. ELISA for Titer Determination and Isotype Analysis. Blood samples from Balb/c mice were allowed stand at room temperature for 3−4 h. Then they were centrifuged at 3000 rpm for 10 min, and the supernatants (serum) were collected and used for following experiments. For IgG titer determination and isotype analysis, we used the same methods as previously described with slight modification.21 Briefly, AβpE3‑15, AβpE3‑40, or Aβ1−40 were dissolved in bicarbonate− carbonate buffer (0.1 M, pH = 9.6) to 20 μg/mL. ELISA plates (96well, Costar 3590) were coated with the solution (100 μL per well) and allowed to stand at 4 °C overnight. After blocking the wells with 0.25% gelatin for 3 h, serially diluted serum was added to the wells, and the plates were incubated at 37 °C for 90 min. The plates were washed with PBS-Tween (0.05% Tween 20, PBS) 3 times. Then, HRP-conjugated rabbit anti-mouse IgG (1:2000, 100 μL per well) was added to the wells for titer determination. For isotype analysis, HRP conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, and IgM (1:1000, 100 μL per well) were added to the wells. After incubating for another 90 min, the plates were washed 3 times again, and substrate was added to the wells. The plates were allowed to stand in a dark place for 20 min, and then OD450 was measured. We define titers as the highest dilution factor where the OD450 minus 0.1 is still higher than the OD450 of negative control serum.22 Morris Water Maze. The Morris water maze (MWM) test was performed when the AD model mice reached 12 months old. We used the methods as described previously19 with some modification. The mice were trained 4 times a day with different starting points. Those who could not find the platform in 1 min would be guided to stay at the platform for 10 s. After 7 days of training, the platform was removed at day 8 and the movements of the mice were recorded and analyzed. EthoVision11.5 was used in this experiment. Brain Collection. After MWM test, the AD model mice were perfused with physiological saline. The brain was separated from body and divided into two hemispheres quickly and carefully. The left hemisphere was frozen in liquid nitrogen and stored at −80 °C, while the right hemisphere was immersed in 4% paraformaldehyde at 4 °C for 24 h. Immunohistochemistry. Immunohistochemistry was performed as previously described23 with slight modification. After paraffin embedding, the right hemisphere were sectioned (5 μm) using a Leica RM2235. After treatment with xylol, the sections were hydrated using different concentrations of ethanol. After antigen retrieval with sodium citrate buffer, the sections were blocked with 10% goat serum for 60 min. Then, 6E10 (BioLegend, 1:200) was incubated with the sections

Figure 4. (A) Sections were immunostained with 6E10, scale bar = 100 μm. (B) Statistical data was collected by ImageJ. Student’s t test was used to calculate p value.

showed less Aβ plaques and better cognitive behavior than the untreated mice, while the mice immunized with Aβ1−15−P2 showed less improvement. In conclusion, our novel prophylactic vaccine, AβpE3‑15−P2, is effective in inducing strong immune response and slowing the progression of AD-like pathology in AD model mice. This new type of vaccine targeting modified Aβ species will open up new directions for the development of active immunotherapy for AD.

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METHODS

Vaccine Preparation. Reagents were purchased from GL Biochem and Sigma-Aldrich unless otherwise stated. All peptides, including AβpE3‑15, Aβ1−15, AβpE3‑40, and peptides for vaccine, were synthesized by Fmoc based solid-phase peptide synthesis and purified by high performance liquid chromatography (Waters-600-2487). BSAAβpE3‑15 conjugate was synthesized by the methods described in our previous work.14 Briefly, we used Fmoc-Lys (ivDde)-Wang resin to synthesize AβpE3‑15-Lys (with all protecting groups) on resin. Then the protecting group ivDde can be selectively removed by 2% N2H4 so that we could add a spacer (triethylene glycol) to the amino group of Lys. After cleaving the peptide from resin, we could get AβpE3‑15-Kspacer. Then we could utilize diethyl squarate to link AβpE3‑15-K-spacer D

DOI: 10.1021/acschemneuro.6b00336 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience for 90 min, after which goat anti-mouse IgG (H + L) antibody conjugated with Alexa Fluor 488 (Invitrogen; 1:200) was incubated with the sections for another 60 min. Finally, Prolong Gold antifade reagent (with DAPI) (Invitrogen) was used to seal the sections. We used laser scanning confocal microscope (Zeiss LSM 780) to observe these sections. Generally, 10× objective was used, and region CA3 was photographed and calculated for plaque quantity.

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pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis. J. Biol. Chem. 285 (53), 41517−41524. (7) Demattos, R. B., Lu, J., Tang, Y., Racke, M. M., Delong, C. A., Tzaferis, J. A., Hole, J. T., Forster, B. M., McDonnell, P. C., Liu, F., Kinley, R. D., Jordan, W. H., and Hutton, M. L. (2012) A plaquespecific antibody clears existing beta-amyloid plaques in Alzheimer’s disease mice. Neuron 76 (5), 908−920. (8) Frost, J. L., Liu, B., Kleinschmidt, M., Schilling, S., Demuth, H. U., and Lemere, C. A. (2012) Passive immunization against pyroglutamate-3 amyloid-beta reduces plaque burden in Alzheimer-like transgenic mice: a pilot study. Neurodegener. Dis. 10 (1−4), 265−270. (9) Acero, G., Manoutcharian, K., Vasilevko, V., Munguia, M. E., Govezensky, T., Coronas, G., Luz-Madrigal, A., Cribbs, D. H., and Gevorkian, G. (2009) Immunodominant epitope and properties of pyroglutamate-modified Abeta-specific antibodies produced in rabbits. J. Neuroimmunol. 213 (1−2), 39−46. (10) Weiner, H. L., and Frenkel, D. (2006) Immunology and immunotherapy of Alzheimer’s disease. Nat. Rev. Immunol. 6 (5), 404− 416. (11) Monsonego, A., and Weiner, H. L. (2003) Immunotherapeutic approaches to Alzheimer’s disease. Science 302 (5646), 834−838. (12) Cai, H., Chen, M. S., Sun, Z. Y., Zhao, Y. F., Kunz, H., and Li, Y. M. (2013) Self-adjuvanting synthetic antitumor vaccines from MUC1 glycopeptides conjugated to T-cell epitopes from tetanus toxoid. Angew. Chem., Int. Ed. 52 (23), 6106−6110. (13) Cribbs, D. H., Ghochikyan, A., Vasilevko, V., Tran, M., Petrushina, I., Sadzikava, N., Babikyan, D., Kesslak, P., KieberEmmons, T., Cotman, C. W., and Agadjanyan, M. G. (2003) Adjuvantdependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int. Immunol. 15 (4), 505−514. (14) Cai, H., Huang, Z. H., Shi, L., Sun, Z. Y., Zhao, Y. F., Kunz, H., and Li, Y. M. (2012) Variation of the Glycosylation Pattern in MUC1 Glycopeptide BSA Vaccines and Its Influence on the Immune Response. Angew. Chem., Int. Ed. 51 (7), 1719−1723. (15) Davtyan, H., Ghochikyan, A., Petrushina, I., Hovakimyan, A., Davtyan, A., Poghosyan, A., Marleau, A. M., Movsesyan, N., Kiyatkin, A., Rasool, S., Larsen, A. K., Madsen, P. J., Wegener, K. M., Ditlevsen, D. K., Cribbs, D. H., Pedersen, L. O., and Agadjanyan, M. G. (2013) Immunogenicity, efficacy, safety, and mechanism of action of epitope vaccine (Lu AF20513) for Alzheimer’s disease: prelude to a clinical trial. J. Neurosci. 33 (11), 4923−4934. (16) Oh, M. M., Simkin, D., and Disterhoft, J. F. (2016) Intrinsic Hippocampal Excitability Changes of Opposite Signs and Different Origins in CA1 and CA3 Pyramidal Neurons Underlie Aging-Related Cognitive Deficits. Front. Syst. Neurosci. 10, 52. (17) Nava-Mesa, M. O., Jimenez-Diaz, L., Yajeya, J., and NavarroLopez, J. D. (2013) Amyloid-beta induces synaptic dysfunction through G protein-gated inwardly rectifying potassium channels in the fimbria-CA3 hippocampal synapse. Front. Cell. Neurosci. 7, 117. (18) Frost, J. L., Le, K. X., Cynis, H., Ekpo, E., Kleinschmidt, M., Palmour, R. M., Ervin, F. R., Snigdha, S., Cotman, C. W., Saido, T. C., Vassar, R. J., St George-Hyslop, P., Ikezu, T., Schilling, S., Demuth, H. U., and Lemere, C. A. (2013) Pyroglutamate-3 amyloid-beta deposition in the brains of humans, non-human primates, canines, and Alzheimer disease-like transgenic mouse models. Am. J. Pathol. 183 (2), 369−381. (19) Zhang, J. Y., Cao, Q., Li, S. W., Lu, X. Y., Zhao, Y. X., Guan, J. S., Chen, J. C., Wu, Q., and Chen, G. Q. (2013) 3-Hydroxybutyrate methyl ester as a potential drug against Alzheimer’s disease via mitochondria protection mechanism. Biomaterials 34 (30), 7552− 7562. (20) (a) Lalonde, R., Kim, H. D., Maxwell, J. A., and Fukuchi, K. (2005) Exploratory activity and spatial learning in 12-month-old APP(695)SWE/co+PS1/DeltaE9 mice with amyloid plaques. Neurosci. Lett. 390 (2), 87−92. (b) Garcia-Alloza, M., Robbins, E. M., ZhangNunes, S. X., Purcell, S. M., Betensky, R. A., Raju, S., Prada, C., Greenberg, S. M., Bacskai, B. J., and Frosch, M. P. (2006) Characterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease. Neurobiol. Dis. 24 (3), 516−524.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00336. Supplementary Figures, Fmoc based solid phase peptide synthesis, HPLC procedures, and characterization of the peptides and protein (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Prof. Dr. Yan-Mei Li. E-Mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Major State Basic Research Development Program of China (Grant 2013CB910700) and the National Natural Science Foundation of China (Grants 21472109 and 91313301). The AD model mice were kind gifts from Prof. Yan-Dao Gong and Prof. Guo-Qiang Chen.

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DOI: 10.1021/acschemneuro.6b00336 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX