Multiple Antigenic Peptide System Coupled with Amyloid Beta Protein

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Multiple antigenic peptide system coupled with amyloid beta protein epitopes as an immunization approach to treat Alzheimer's disease Diya Sun, Yongbo Qiao, Xiaoyu Jiang, Pengju Li, Ziyu Kuai, Xin Gong, Dongni Liu, Qiang Fu, Liyan Sun, He Li, Jun Ding, Yuhua Shi, Wei Kong, and Yaming Shan ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/ acschemneuro.9b00020 • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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ACS Chemical Neuroscience

Multiple antigenic peptide system coupled with amyloid beta protein epitopes as an immunization approach to treat Alzheimer's disease Diya Sun,[1] Yongbo Qiao,[1] Xiaoyu Jiang,[1] Pengju Li, [1] Ziyu Kuai,[1] Xin Gong,[1] Dongni Liu,[1] Qiang Fu,[2] Liyan Sun,[3] He Li,[4] Jun Ding,*[5] Yuhua Shi,[1] Wei Kong,[1] Yaming Shan*[1]

Abstract Latest studies suggest that Alzheimer's disease (AD) is one of the "four big killers" that threaten the health of the elderly. AD affects about 46 million people across the world, and there is a critical need for research on new therapies for treating AD. The purpose of present study was to develop and evaluate immunogens to elicit antibodies against the formation of amyloid beta protein (Aβ), a pathological hallmark of AD, as a therapeutic strategy in AD. In this study, serial potential immunogenic epitopes were screened according to the Aβ sequence. The screened linear epitopes on the Aβ Cterminal fragment were coupled with either the carrier protein keyhole limpet hemocyanin (KLH) or the synthesized 4-branchpeptide (MAP4). MAP4 immunogens could effectively elicit immunogenicity against Aβ1-42 monomer and fiber in Balb/C

Diya Sun (DYS), Yongbo Qiao (YBQ), Xiaoyu Jiang (XYJ), Pengju Li (PJL), Ziyu Kuai (ZYK), Dr. Xin Gong (XG), Dongni Liu (DNL), Yuhua Shi (YHS), Prof. Wei Kong (WK), Prof. Yaming Shan* (YMS) National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, Jilin, China. E-mail: [email protected] 2 Qiang Fu (QF) Affiliated Zhongshan Hospital of Dalian University, Dalian, Liaoning, China. 3 Liyan Sun (LYS) Dalian University Affiliated Xinhua Hospital, Dalian, Liaoning, China. 4 He Li (HL) Affiliated Dalian Friendship Hospital of Dalian Medical University, Dalian, Liaoning, China. 5 Dr. Jun Ding* (JD) China-Japan Union Hospital of Jilin University, Changchun, Jilin, China. E-mail: [email protected] * Corresponding Authors 1

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mice. Furthermore, MAP4 sera could also effectively inhibit the formation of the Aβ142

fiber. Interestingly, one of the MAP4 sera was able to depolymerize the Aβ1-42 fibers

that had aggregated. The monoclonal antibody, 1H7, was shown to inhibit the formation of Aβ1-42 fiber. MAP4 carrier may provide benefits over current immunization strategies, as it does not induce inflammation. In conclusion the MAP4-Aβ conjugates offer a promising approach for the development of a safe and effective AD vaccine.

Key words: Alzheimer’s disease; amyloid beta protein; Aβ fiber depolymerization; multiple antigen peptide system; vaccine candidate; 6E10

Introduction Alzheimer's disease (AD), a neurodegenerative disease, is characterized by the impairment of memory and cognition. It is the number one cause of dementia with roughly 40 million people with AD worldwide and is expected to affect about 135 million by 2050.1,2 Amyloid plaques, consisting of insoluble fibrils of the amyloid-beta protein (Aβ), accumulate in the extracellular matrix.3,4 These amyloid plaque deposits are a major pathological hallmark of AD and are thought to induce dementia.5 Numerous experimental studies demonstrate the critical need to research and develop drugs for the treatment of AD.6-8 Currently the only therapeutics available only relieve temporarily some AD symptoms and among the therapies being developed, Aβ immunization appears to be the most promising.9 Therapy targeting Aβ is a potential approach to attenuate cerebral amyloid plaques.10 Prior research in AD transgenic mouse, treated with active and


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passive immunization, provided evidence that antibodies can reduce amyloid load and the cognitive deficits.11 Although, in earlier 2018, Pfizer unfortunately decided to halt the development of drugs for AD, recent studies that target Aβ have provided promising results. Sodium oligomannurarate (GV-971) capsule from Shanghai's Green Valley Pharmaceutical, a compound that interferes with Aβ aggregation, has met its primary endpoint in the latest Phase 3 clinical trials in patients with mild to moderate AD. Also, the monoclonal antibody BAN2401, developed by Biogen EISAI, was shown to decrease significantly Aβ load in the brain and to slow cognitive decline in patients with mild AD who received 10 mg/kg of the drug twice a week for 18 months. 12,14 Active Aβ immunotherapy can induce effective Aβ antibody titers and has emerged as the most promising and cost-effective approach in AD therapy. However, the clinical trial for an Aβ vaccine, AN1792, was interrupted due to the development of meningoencephalitis in 6% of the patients, likely involving pro-inflammatory CD4+ T cells.15Research studies in 133 patients demonstrated that abundant DR(allele) haplotypes were associated with the Aβ-specific T cell responses, elicited via distinct T cell epitopes on Aβ (residues 15-42).16 CD4+ T cell epitopes are primarily identified in the Aβ15–42 peptide, distinct from the dominant B cell epitopes identified in Aβ1–15.17, 18

Fu and colleagues using chimeric P particles carrying Aβ1-6 as the epitope, blocked

the toxicity of Aβ aggregation in vitro, without inducing the T-cell-mediated immune response.19 In another study, an immunogen consisting of 16 copies of the Aβ1-15 peptide on a lysine backbone, successfully induced Aβ-specific antibodies in an APPtransgenic mice mouse model without producing any cellular immune response to the



antigen.20 These findings indicate that Aβ1-6 and Aβ1-15 continue to be potential epitopes for the development of an AD vaccine. These achieved exciting results have encouraged the vaccine route to prevent AD, although there are some clinical setbacks.21 More and more AD vaccine candidates are constantly appearing.22,23 Most recent studies indicated that Aβ3-10-KLH could increase the level of anti-Aβ immune response, and lead to sustained prevention of Aβ deposition four months after stopping the vaccine treatment.24,25 An important goal for an effective AD vaccine is to produce a high titer of specific antibodies that inhibit fibril formation or depolymerize fibers. Due to the low immunogenicity of the Aβ1-6 or Aβ1-15 epitope, using an effective carrier is imperative to induce a strong immune response for effective AD therapy. Multiple antigenic peptide system (MAPs) is a part of the covalent peptide dendrimers synthesized by Tam and can enhance immunogenicity.26 A MAP molecule contains two functional structures, an inner oligo lysine core and multiple copies of the synthetic antigenic peptide immunogens.26-28 Further, the oligo-lysine cores, with 1-4 sequential levels of lysine residues, can produce different copies of synthetic immunogens.29 Compared with the virus vector vaccines, the artificial molecular vaccine with the lysine core, might effectively avoid the inherent human immune response and improve the effectiveness of the vaccine. Therefore, in this study serial potential immunogenic linear sequences were first screened, following synthesized immunogens with Aβ1-6 or Aβ1-15epitopes by MAPs. Furthermore, the serum activities against amyloid beta protein would be evaluated.

Results and Discussion


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Immunogen synthesis: As MAPs containing gp41 membrane-proximal external region could elicit broad neutralizing antibodies against human immunodeficiency virus type 1 in guinea pigsinpreviousstudy,30 two kinds of novel MAP4 immunogens were designed in this study, one carrying sequence (Aβ1-6)2, and the other carrying sequence Aβ1-15. Additionally, 3 immunogens coupled with carrier protein keyhole limpet hemocyanin (KLH) were also employed. The Aβ1-6, (Aβ1-6)2 and Aβ1-15sequences were coupled with the carrier protein KLH and synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China). Additionally, MAP4immunogens were synthesized by Chinese Peptide Co., Ltd (Shanghai, China). The synthesized sequences are listed in Table 1. Table 1 Synthesis of the Immunogens Immunogens

Sequences

Aβ1-42

Ac-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-NH2

Aβ1-6-KLH

Ac-DAEFRH-C-KLH

(Aβ1-6)2-KLH

Ac-DAEFRHDAEFRH-C-KLH

Aβ1-15-KLH

Ac-DAEFRHDSGYEVHHQ-C-KLH[a]

(Aβ1-6)2-MAP4

Ac-DAEFRHDAEFRHKKK-6-Ahx-MAP4 [b]

Aβ1-15-MAP4

Ac-DAEFRHDSGYEVHHQKKK-6-Ahx-MAP4

[a]

The C-terminal of the sequence was connected with KLH, via Cysteine residue(C).

[b]The

C-terminal of the sequence was connected with the poly-lysine core, via a 6-aminohexanoic

acid residue(6-Ahx), to enhance the conformational flexibility of the epitope.

Immunogenicity of the antigens: The immunogenicity evaluation of the antigens was performed in Balb/c mice. The mice were administrated Aβ1-6-KLH, (Aβ1-6)2-KLH, Aβ1-15-KLH, Aβ1-15-MAP4 or (Aβ1-6)2-MAP4, and the antibody titer of serum was determined by ELISA. PBS and



Aβ1-42 were set as the control groups. (Aβ1-6)2-KLH group did not give a strong immunogenic response compared to the Aβ1-6-KLH group. Although a slightly higher serum titer could be induced in the Aβ16-KLH

group, there was no significant difference between them. In Aβ1-15-KLH group,

the serum antibody titer was significantly different when compared with MAP4 immunogens. While, the strongest immunogenicity was elicited by (Aβ1-6)2-MAP4, whose titer reached over 10,000 (Figure 1).

Figure 1 Aβ1-42 specific antibodies in serum using ELISA. The highest reciprocal serum dilution that yielded an absorbance more than twice over the background values was calculated as the ELISA endpoint titer. Comparison of the Aβ binding activities of the antiserum responses induced by various immunogens in Balb/c mice. Serum antibody titers of mice immunized with Aβ1-6-KLH, (Aβ1-6)2-KLH, Aβ1-15-KLH, Aβ1-15-MAP4, or (Aβ1-6)2-MAP4, after a 4-round immunization, were determined by ELISA. *=P

Multiple Antigenic Peptide System Coupled with Amyloid Beta Protein

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