Single Dose of Protein Vaccine with Peptide Nanofibers As Adjuvants

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Single dose of protein vaccine with peptide nanofibers as adjuvants elicits long-lasting antibody titer Chengbiao Yang, Fang Shi, Can Li, Youzhi Wang, Ling Wang, and Zhimou Yang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00488 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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ACS Biomaterials Science & Engineering

Single dose of protein vaccine with peptide nanofibers as adjuvants elicits long-lasting antibody titer Chengbiao Yang, †, # Fang Shi, # Can Li, # Youzhi Wang, # Ling Wang, *, † Zhimou Yang*, #

†

State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key

Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, P. R. China #

Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai

University, and Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071, P. R. China

Email: [email protected] and [email protected]

ACS Paragon Plus Environment

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ABSTRACT Self-assembling materials based on peptides have shown great potential as vaccine adjuvants. In our previous work, we have demonstrated that nanofibers based on D-peptide NapGDFDFDY are good candidates for vaccine adjuvants. Here we further found that supramolecular hydrogels based on positively charged D-peptide Nap-GDFDFDYDK as vaccine adjuvants could induce stronger immune response. We designed and synthesized two D-peptide derivatives, one with a positive charge (Nap-GDFDFDYDK) and the other with a negative charge (NapGDFDFDYDE). Both of them could form the hydrogels constructed by nanofibers. The nanofibers formed by Nap-GDFDFDYDK promoted the more powerful immune response in mice against the antigen chicken egg albumin (OVA) than peptides Nap-GDFDFDY and Nap-GDFDFDYDE. Through cell experiments, we demonstrated that the main reason was that nanofibers formed by Nap-GDFDFDYDK could enhance the uptake of OVA by primary antigen presenting cells. Most importantly, it was intriguing that the nanofibers based on Nap-GDFDFDYDK could evoke longlasting antibody titers for 28 weeks at a single dose of protein vaccine. Our study demonstrated that supramolecular hydrogels based on positively charged D-peptide were promising vaccine adjuvants and might be very useful for antibody production and vaccine development.

Keywords: Self-assembly; Vaccine adjuvant; Peptide; Nanofiber


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1. INTRODUCTION Vaccine has been a kind of potential treatment for diverse diseases such as contagion,1-3 cancer,4-7 and diabetes.8-13 Particularly, the subunit vaccines have attracted more and more interests due to their well-defined molecular structures, safety, and antigenicity compared with attenuated live vaccines. However, their applications in clinic were limited by their poor immunogenicity because of their rapid clearance and poor delivery efficiency to the target in the host. Aiming to improve the immunogenicity, researchers have developed a variety of immune adjuvants such as aluminum salt precipitates (Alum),14-15 toll-like receptors (TLR) agonists,16-20 and polymers.21-26 However, the efficiency and biosafety of these materials are insufficient yet to prevent and treat some obstinate diseases such as HIV and cancers. Therefore, it is necessary to develop new generation adjuvants with good biocompatibility, biodegradability, lower immunogenicity, and outstanding capability to induce an immune response against the antigen. In the past decades, self-assembling materials27 based on peptides have shown great potential in the field of regenerative medicine,28-30 drug delivery,31-37 cell culture,38-41 and detection of bioactive molecules42-45 due to their ease of design, synthesis, and incorporation with functional motifs, biocompatibility, and low immunogenicity. Nevertheless, their application as vaccine adjuvants has been explored rarely. Recently, Collier and co-workers have used self-assembling peptides as powerful immune adjuvants to boost the immunogenicity of the epitope from chicken egg albumin (OVA) which could raise antibody responses in mice.46-47 Li group reported that self-adjuvanting MUC1 glycopeptide vaccine candidates could provoke the cellular immune response to suppress the proliferation of cancer cell.48-49 Tirrell and co-workers also developed a kind of micelle constructed by peptide amphiphiles which served as an effective antigen delivery system.50-51 Although these strategies could dramatically enhance the immune


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response, there was a prerequisite that the protein or epitope was covalently conjugated with the self-assembling peptides. In order to make it easy-to-use, blending the materials with antigen physically is a promising alternative.

Scheme1.The chemical structures of compounds and the optical picture of hydrogels (0.5 wt %).

In our previous works, we discovered a supramolecular hydrogel formed through enzymatic catalysis, could serve as a promising vaccine adjuvant.52 We subsequently found that a supramolecular hydrogel based on D-peptide derivative Nap-GDFDFDY (named as Y) acquired by the heating-cooling method also promoted the activation of CD8+ IFN-γ+ T cells.53 All of the adjuvants used in these assays were simply mixed with antigens physically. We herein introduce an additional amino acid with different charges (DK or DE) at the carboxyl terminal of the peptide Y to form the peptide Nap-GDFDFDYDK and Nap-GDFDFDYDE (Scheme 1, named as YK, YE respectively). We postulate that they may further augment the immune response against the antigen and improve the efficiency of therapeutic and prophylactic vaccines. We found that the more positively charged nanofibers formed by YK could enhance the antigen uptake by primary antigen presenting cells (DCs), resulting in promoting the more powerful immune response against the antigen than peptide Y and YE. In addition, it was intriguing that the nanofibers


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based on YK could evoke long-lasting antibody titers for 28 weeks at a single dose of protein vaccine.

2. MATERIALS AND METHODS Materials: All Fmoc-amino acids were purchased from GL Biochem (Shanghai). EndoFit Ovalbumin (endotoxins < 1EU/mg) was obtained from InvivoGen (CA, USA). Recombinant mouse GM-CSF and IL-4 were purchased from Peprotech (Rocky Hill, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b were acquired from Southern Biotechnologies (AL, USA). Mouse IL-6 ELISA kits were obtained from Biolegend (CA, USA). Alum adjuvant was bought from Pierce Biotechnology (IL, USA). All mice were used at 6-8 weeks old and strictly according to the related rule of Animal ethics association. General methods: 1H NMR (Bruker ARX 400) and ESI-MS (LCQ ADMAX) were used to characterize the synthesized compounds. Preparation of peptides: Peptide Nap-GDFDFDY, Nap-GDFDFDYDK, and Nap-GDFDFDYDE were prepared by solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding N-Fmoc protected amino acids with side chains properly protected by a tert-butyl group. The first amino acid (Fmoc-D-Tyr(OtBu)-OH, Fmoc-D-Lys(Boc)-OH or Fmoc-DGlu(OtBu)-OH) was loaded on the resin at the C-terminal with the loading efficiency about 1.0 mmol/g. 20% of piperidine in anhydrous N, N’-dimethylformamide (DMF) was used to remove Fmoc group. Then the next Fmoc-protected amino acid was coupled to the free amino group using O-(Benzotriazol-1-yl)-N, N, N’, N’-tetramethyluronium hexafluorophosphate (HBTU) as


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the coupling reagent. The growth of the peptide chain was according to the established Fmoc SPPS protocol. At the final step, Naphthalene (Nap) group was used to couple with the peptide. After the last coupling step, excessive reagents were removed by a single DMF wash for 5 min (5 ml per gram of resin), followed by five steps of washing using dichloromethane (DCM) for 2 min (5 ml per gram of resins). The peptide derivatives were cleaved from the resin by ice-cold reagent B (95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% double-distilled water), and then the mixture was stirred at room temperature for 30 min, filtered, and poured into icecold diethylether. The resulting precipitate was centrifuged for 10 min at 4℃ at 10,000 rpm. Afterward the supernatant was decanted and dissolved in double-distilled water and lyophilized. The crude product was further purified by HPLC and dried by a lyophilizer. Preparation of hydrogels: (1) 1 mg of Nap-GDFDFDY or Nap-GDFDFDYDE and 1 equv. of Na2CO3 (to adjust the pH to 7.4) were dissolved in 0.2 mL of 1×PBS (pH=7.4) at room temperature. Then the solution was heated to boil. The hydrogel was formed after cooling at room temperature for 25 min. (2) 1 mg of Nap-GDFDFDYDK were dissolved in 0.1 mL of doubledistilled water and heated to boil to make them dissolve. And then 0.1 mL of 2×PBS containing 1 equv. of Na2CO3 (to adjust the pH to 7.4) were added immediately, the hydrogel was acquired after cooling at room temperature for 5 min. The method to prepare the hydrogel of NapGDFDFDYDK is different because its solubility in water is poor. Transmission electron microscopy (TEM): TEM samples were prepared as follow. A copper coated with a thin layer of carbon layer was dipped into the solution of YE or YK (0.2 wt %) with or without OVA and then it was kept in a desiccator overnight and later negatively stained


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with uranyl acetate. The dried sample was performed at the Tecnai G2 F20 system, operating at 200 kV. Biocompatibility assessment of nanofibers: The biocompatibility of the nanofibers was evaluated by the CCK-8 assay. The RAW 264.7 cells and splenocytes from mouse spleen were seeded in 96-well plates at a density of 10,000 cells per well with a total medium volume of 200 µL and incubated for 24 hours. Then the media were removed and 200 µL of the solutions containing a serial of concentrations of the self-assembling nanofibers, which were in solution state, were added into the cells. 72 hours later, 10 µL of CCK-8 per well were added. After 4 hours, the optical density of the medium containing CCK-8 at 597 nm was measured by using a microplate reader (Bio-RADiMarkTM, America). Cells without any treatment were used as the control. The cell viability percent was calculated according to the following formula: The cell viability percent (%) = ODsample/ODcontrol×100%. Preparation of different formulation of vaccines: For three vaccine groups which used nanofibers as adjuvants, 30 µL of OVA or FITC-OVA (5 mg/mL, endotoxin-free) in PBS buffer was added into 300 µL of hydrogel (5 mg/mL), and then the mixture was diluted with PBS into 750 µL of the solution. The resulting mixture was mixed homogeneously through vortex. For the alum adjuvant group (Al group), 75 µL of Alum adjuvant licensed by FDA (40 mg/mL) were blended with 24 µL of solute OVA (5 mg/mL, endotoxin-free) physically, and then diluted with PBS into 600 µL of the solution. For the control group (OVA group), 0.2 mg/mL of solute OVA or FITC-OVA in PBS was used. All the groups are in the solution state and the final concentration of OVA or FITC-OVA was 0.2 mg/mL in all groups.


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FITC-OVA uptake by DCs: Bone marrow cells were isolated from C57/ BL6J mouse femur and tibia, and then cultured in X-vivo 15 medium (Lanza, MD, USA) containing GM-CSF (20 ng/ml) and IL-4 (10 ng/ml) at 37 °C for 6 days to acquire immature dendritic cells (DCs). The obtained DCs were incubated with 6.25 µL of different vaccine groups containing 1.25 µg of FITC-OVA for 0.5 h. And the same amount of FITC-OVA was used as the control. The assay of uptake was performed by flow cytometer. The test of cytokines: The immature DCs were stimulated with 50 µL of different vaccine groups (0.2 wt %) for 24 h. At the end of the experiment, the supernatant was harvested by using ELISA kits (Biolegend, SanDiego, CA). Vaccination: C57BL/6 mice were used at 6-8 weeks old. All the above vaccine groups in the solution state were injected subcutaneously at a final volume of 100 µL ([OVA] = 0.2 mg/mL) on day 0, 14 or only on day 0. Titer measurement: High-binding ELISA plates (Costar 3590) were coated with 10 µg/ml of OVA in coating buffer overnight at 4°C. The plates were washed 3 times by PBST (0.1% Tween-20 in PBS). After blocked with 1% BSA in PBST for 1 h, the serum was diluted 2 times and incubated in each well for 2 hours at room temperature. Horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, IgG2a, or IgG2b was diluted to working concentration and incubated (100 µL per well) for 1h at room temperature after washed three times. After washed five times, 100 µL of TMB Substrate was added to per well. After 30 min incubation at room temperature, H2SO4 solution was added to end the reaction. Optical absorption was measured at 450nm. The highest dilution, which yields an optical absorption of 0.1 or greater, is calculated as efficient titer.


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3. RESULTS AND DISCUSSION

Figure 1. TEM images of the self-assembling materials A) Nap-GDFDFDYDE (0.2 wt %); B) Nap-GDFDFDYDE (0.2 wt %) with OVA ([OVA] = 0.2 mg/mL); C) Nap-GDFDFDYDK (0.2 wt %); and D) Nap-GDFDFDYDK (0.2 wt %) with OVA ([OVA] = 0.2 mg/mL). The scale bars represent 1 µm.

Characterization of nanofibers with or without OVA. It was shown that both of NapGDFDFDYDE (YE) and Nap-GDFDFDYDK (YK) could form hydrogels at the concentration of 0.5wt% by the heating-cooling method (scheme 1). The hydrogel formed by YE was transparent, whereas the hydrogel formed by YK was translucent. However, after blending with OVA physically, both of the hydrogels were broken and changed to solutions. We also found that below 0.5wt %, YE or YK cannot form the hydrogel (for example, at 0.2wt %). Therefore, TEM assay was performed to characterize the microtopography of the solution of YE or YK with or without OVA. As shown in Figures 1A and 1C, both of YE and YK self-assembled to nanofibers. It was observed that the nanofibers formed by YE were pliable and entangled with


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each other, while the nanofibers constructed by YK were shorter and thicker. The features of self-assembling nanofibers based on both peptides were not affected after mixed with OVA (Figures 1B and 1D), suggesting that protein OVA was physically adhered at the surface of nanofibers or existed in the cavity of three dimensional fiber networks. The ability of nanofibers to promote the immune response. Prior to evaluate the capability of nanofibers to stimulate the immune response against antigen, the biocompatibility of the selfassembling peptides was assessed by the CCK-8 assay. It was obvious that both YK and YE had good biocompatibility (Figure S5). We then compared the vaccine adjuvant potency of our peptide hydrogels. C57BL/6 mice were vaccinated subcutaneously with different formulations. The nanofibers based on YK, YE, and Y were blended with OVA physically to form three experimental groups, called as YK group, YE group and Y group respectively. The Alum adjuvant licensed by FDA containing OVA (Al group) and Y group were set as the positive controls. The PBS solution of OVA (OVA group) was used as a negative control. It was obvious that all the nanofibers as adjuvants could induce stronger immune responses against antigen compared with the OVA itself. Compared with Al group, only YK and Y group showed signiﬁcantly higher anti-OVA IgG antibody productions (Figures 2A-2D). For the YK group, it triggered the strongest anti-OVA immune response than other groups, and it increased IgG production by 260, 4.33, 2.60, and 6.71-fold to OVA, Al, Y, and YE group, respectively. These results indicated that the co-assembled supramolecular nanoﬁbers could strongly enhance the immune responses in vivo, possibly due to their capability of controlled release of antigen.54 Compared with YK and Y group, YE group showed lower immune potency because OVA antigen is negative charged, negatively charged nanofibers of YE might hinder it from coassembling with OVA antigen. On the contrary, due to carrying the positive charges, YK


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adjuvant could co-assemble well with OVA antigen, thereby stimulating the excellent immune responses against antigen. Pioneering research also indicated that positively charged nanomaterials showed better cellular uptakes.55 Therefore, our nanofibers of YK might enhance the uptake of OVA by antigen presenting cells (APCs).

Figure 2. The effect of YK and YE as adjuvants on OVA-specific antibodies production: A) IgG, B) IgG1, C) IgG2a, and D) IgG2b. (Alum, Y as adjuvants and free OVA were used as controls). E) Schematic graph of immune study. The data were shown as mean ± standard deviation (n = 5) and analysed by unpaired t-test (* p

Single Dose of Protein Vaccine with Peptide Nanofibers As Adjuvants

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