Cell Penetrating Peptides-based Redox-sensitive Vaccine Delivery

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Cell penetrating peptides-based redox-sensitive vaccine delivery system for subcutaneous vaccination Kewei Wang, Yong Yang, Wei Xue, and Zonghua Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00905 • Publication Date (Web): 23 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Molecular Pharmaceutics

Cell Penetrating Peptides-based Redox-sensitive Vaccine Delivery System for Subcutaneous Vaccination

Kewei Wang, Yong Yang, Wei Xue, Zonghua Liu * Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou, 510632, China

* Corresponding author: Zonghua Liu ([email protected])

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract:

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In immunotherapy, induction of potent cellular immunity by vaccination is essential to

treat intracellular infectious diseases and tumors. In this work, we designed a new synthetic peptide carrier

Cys-Trp-Trp-Arg8-Cys-Arg8-Cys-Arg8-Cys

for

vaccine

delivery

by

integrating

redox-responsive disulfide bond cross-linking and cell-penetrating peptide Arginine octamer. The carrier peptide bound to antigen protein ovalbumin (OVA) via electrostatic self-assembly to form peptide/OVA nanocomposites. Then, the spontaneous oxidization of the thiols of the cysteine residues induced interpeptide disulfide bond crosslinking to construct denser peptide/OVA condensates. The cell-penetrating peptides incorporated in the carrier peptide could increase antigen uptake by antigen presenting cells. After internalized by antigen presenting cells, the antigen could be rapidly released in cytoplasm along with degradation of the disulfide bonds by intracellular glutathione, which could promote potent CD8+ T cell immunity. The cross-linked peptide/OVA condensates were used for subcutaneous vaccination. The results showed that, the peptide carrier mediated potent antigen-specific immune response by significantly increasing IgG titer, splenocyte proliferation, secretion level of cytokines INF-γ, IL-12, IL-4 and IL-10, immune memory function, and activation and maturation of dendritic cells. From the results, the low molecular weight vaccine condensing peptide with definite chemical composition could be developed as a novel class of vaccine delivery systems.

Key words: vaccine delivery system, redox-responsive, cell-penetrating peptides, cellular immunity


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1 INTRODUCTION In immunotherapy, induction of potent cellular immunity by vaccination is essential to treat intracellular infectious diseases and tumors. Currently, traditional vaccine adjuvants such as aluminium adjuvant and Freund’s adjuvant (FA) mainly help antigen to induce humoral immunity. However, humoral immunity could not effectively fight against those intracellular infectious diseases and tumors. In cellular immunity, the produced CD8+ T cells could directly kill virus-infected cells or cancerous cells. Therefore, induction of potent cellular immunity has gained more and more attention in the immunotherapy of intracellular infectious diseases and tumors. To induce potent cellular immunity, various strategies have been adopted, such as using pH-labile vaccine carrier materials to achieve endosomal escape,1-8 and using redox-responsive vaccine carrier materials to obtain cytoplasmic delivery of antigens.9-15 In this work, to induce potent cellular immunity, we designed a new synthetic peptide for vaccine delivery by integrating redox-responsive disulfide bond cross-linking and cell-penetrating peptides. Cell-penetrating peptides are a type of oligopeptides that could carry macromolecule cargos to cross cell membrane in a membrane-friendly way,16 and hence are ideal carriers for intracellular delivery. In the field of gene therapy, cell-penetrating peptides have been widely used to achieve cytosolic delivery of siRNAs in target cells.17 In the field of vaccination immunotherapy, cell-penetrating peptides have also been used to achieve intracellular delivery of antigens into antigen presenting cells (APCs).18,

19

In these studies, cell-penetrating peptides were physically

bound or covalently conjugated to antigen molecules, which could increase antigen uptake by antigen presenting cells and further enhance antigen-specific immune response. To promote cross-presentation and cellular immunity, redox-responsive vaccine carrier materials have been used to obtain cytoplasmic delivery of antigens. Generally, these redox-responsive vaccine carrier materials contain redox-responsive disulfide bonds that can be degraded by intracellular glutathione. Along with the reduction of disulfide bonds, antigen molecules could be release from the carrier materials to cytoplasm, and could be cross-presented to induce cellular immunity. In these redox-responsive vaccine carrier materials, disulfide bonds could be introduced by using the polymers bearing disulfide bonds in the main chains,9 using the



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polymers bearing disulfide bonds in the side chains,10 using cross-linking agents containing disulfide bonds,11,

12

or attaching antigen molecules or antigen peptides to carrier materials by

disulfide bonds.13-15 In this work, the advantages of cell-penetrating peptides and redox-responsive disulfide bond cross-linking

were

combined

to

design

a

vaccine

carrier

peptide

Cys-Trp-Trp-Arg8-Cys-Arg8-Cys-Arg8-Cys for antigen delivery. Compared with those carrier polymer materials, the carrier peptide has definite molecular weight and chemical composition, can be synthesized in a controlled manner, and hence has good reproducibility between different batches. Moreover, the carrier peptide with abundant positive charges would bind to negatively charged antigen proteins via electrostatic attraction to form nanocomposites, which is like a cage to encapsulate antigen molecules. In the nanocomposites, the thiols of the cysteine residues undergo spontaneous oxidization to form interpeptide disulfide bond crosslinking to construct denser peptide/antigen condensates. After vaccinating with the redox-responsive peptide/antigen condensates containing cell-penetrating peptides, the elicited antigen-specific immune responses were evaluated in this work.

2 EXPERIMENTAL 2.1 Materials The peptide (CWWRRRRRRRRCRRRRRRRRCRRRRRRRRC) was synthesized by GL Biochem Ltd (Shanghai, China). Ovalbumin (OVA) and Freund’s adjuvants were provided by Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 medium and fetal bovine serum were obtained from Gibco (Carlsbad, CA, USA). Cell counting kit-8 (CCK-8) was obtained from Dojindo (Japan). All mouse enzyme-linked immunosorbent assays (ELISA), anti-mouse antibodies and fluorescent labeled anti-mouse antibodies were obtained from Biolegend (San Diego, CA, USA). ELISpotPLUS kits were purchased from Mabtech AB (Nacka Strand, Sweden). Female Balb/c mice of 4-6 weeks were provided by Beijing HFK Bio-Tech Company (Beijing, China). 2.2 Preparation and characterization of the peptide/OVA nanocomposites


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The peptide/OVA nanocomposites were prepared by mixing the same volume of 300 µg/mL of the peptide solution with 600 µg/mL of OVA solution, and then incubating the resulting suspension for 2 h at room temperature to allow disulfide cross-linking. The size and zeta potential of the obtained peptide/OVA nanocomposites were measured by using Nano laser particle size analyzer (Zetasizer Nano ZS, Malvern, UK). 2.3 Redox-sensitivity of the peptide/OVA nanocomposites To investigate the redox-sensitivity of the peptide/OVA nanocomposites, dithiothreitol (DTT) was added into the suspension of the peptide/OVA nanocomposites to a final concentration of 10 mM. The absorbance of the suspension at 500 nm was measured at different times by using a UV spectrophotometer (UV-2550, Suzhou Shimadzu Corporation, China). In addition, the size of the nanocomposites was measured before and after the DTT treatment. 2.4 Antigen uptake and intracellular localization in DC2.4 cells DC2.4 cells in DMEM complete medium were seeded and cultured for 24 h in a 24-well plate with the density of 1×105 cells/well. After removing the medium, the cells were rinsed and then incubated for 4 h with OVA-Cy5.5 solution or the peptide/OVA-Cy5.5 suspension. The concentration of OVA-Cy5.5 in the OVA-Cy5.5 solution or the peptide/OVA-Cy5.5 suspension was 5 µg/mL. Then, the cells were washed and analyzed with flow cytometry (cytoFLEX, Beckman, USA). To track antigen intracellular localization, DC2.4 cells in DMEM complete medium were cultured for 1 d in a poly-D-lysine-coated Petri dish with the density of 1×105 cells/well. After removing the medium, the cells were rinsed and then incubated for 4 h with OVA-Cy5.5 solution or the peptide/OVA-Cy5.5 suspension. The concentration of OVA-Cy5.5 in the OVA-Cy5.5 solution or the peptide/OVA-Cy5.5 suspension was 5 µg/mL. Then, the cells were washed and incubated with LysoTracker-green DND-99 (Molecular Probes-Invitrogen, CA, USA) to label the lysosomes. Then, the cells were viewed with a confocal laser scanning microscope (TCS SP5, Leica, Germany). 2.5 Immunohistochemistry assay



The mice were randomly grouped (n=4) for subcutaneous vaccination. Each group was injected with 100 µL (50 µL/hind leg) of the antigen formulations as listed in Table 1. At 2 or 7 d after the immunization, two mice of each group were euthanized. The spleens were separated and fixed with 4% paraformaldehyde for immunohistochemistry assay. 2.6 Subcutaneous vaccination The mice were randomly grouped (n=5) for subcutaneous vaccination. Each group was injected with 100 µL (50 µL/hind leg) of the antigen formulations as listed in Table 1. The mice were immunized three times with an interval of 7 days. At 8th day after the third immunization, blood was obtained from the mice. The sera was prepared and stored at -20°C for further measurement. Splenocytes were collected from the spleens of the immunized mice for further analysis. 2.7 Titer determination of antigen-specific serum IgG and IgG subtypes The titer of antigen-specific serum IgG and IgG subtypes were measured by ELISA. Briefly, 96-well ELISA plates were treated at 4°C for 16 h with 100 µL coating buffer (0.05 M carbonate buffer solution, 10 µg/mL OVA, pH9.6). After that, the plates were rinsed thrice with 200 µL PBST (PBS containing 0.05% Tween-20). Then, 200 µL blocking solution (PBST containing 2% m/v bovine serum albumin) was pipetted to each well for 1 h of incubation at 37°C under shaking. After washing the plates thrice with 200 µL PBST, 100 µL of diluted serum was pipetted into each well for 1 h of incubation at 37°C under shaking. After washing the plates, 50 µL horseradish peroxidase-conjugated goat antibodies against either mouse IgG, IgG1, or IgG2a (diluted 1:8000 in PBST) were pipetted to each well for 30 min of incubation at 37°C. After washing the plates, 100 µL of 3,30,5,50-tetramethylbenzidine (TMB) substrate was pipetted to each well for 10 min of incubation at room temperature in dark. The color-reaction was terminated by pipetting 100 µL of 2 M H2SO4 to each well. The absorbance at 450 nm of each well was read with a microplate reader (Thermo Fisher Scientific, USA). 2.8 Splenocyte proliferation


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Splenocyte suspension (100 µL) was seeded and cultured in 96-well plates at the density of 5×105 cells/well for 72 h of stimulation by OVA solution (100 µL, 20 µg/mL) or not. After that, 20 µL of CCK-8 reagent was pipetted into each well for 4 h of incubation. The absorbance at 450 nm of each well was read with the microplate reader. The proliferation index (PI) was calculated by the following equation:

PI =

OD2 − OD0 OD1 − OD0

Where OD0, OD1 and OD2 were the absorbance values of blank cultures, non-stimulated cultures and stimulated cultures, respectively. 2.9 Determination of cytokine levels by ELISA assay Splenocyte suspension (2 mL) was seeded and cultured in 12-well plates at the density of 1×107 cells/well for 60 h of stimulation by OVA solution (2 mL, 20 µg/mL in RPMI 1640 medium). After that, the supernatants were collected. The levels of IFN-γ, IL-12, IL-4 and IL-10 were assayed by using ELISA MAXTM Deluxe Sets in accordance with the manufacturer’s instructions. 2.10 Determination of the frequency of the IFN-γ or IL-12 secreting spenocytes by ELISpot assay Briefly, 96-well ELISpot plates were treated for 30 min at room temperature with 200 µl RPMI 1640 medium (containing 10% fetal bovine serum). Then, splenocyte suspension (100 µL, 4×105 cells/well) was added into each well and stimulated with OVA solution (100 µL, 20 µg/mL in RPMI 1640 medium). The IFN-γ or IL-4 secreting cells was detected by using mouse ELISpot PLUS kits in accordance with the manufacturer’s instructions. 2.11 Determination of OVA-specific memory T cells The splenocytes cultured in 24-well plates were subject to 60 h of stimulation by OVA solution (10 µg/mL). After that, the cells were collected, rinsed, and then stained for 30 min at 4°C in dark with the fluorochrome-conjugated anti-mouse antibodies: FITC-anti-CD4, Cy5.5-anti-CD8a, PE-anti-CD44, and APC-anti-CD62L. After the staining process, the splenocytes were examined by flow cytometry.



2.12 Expression of MHC and the co-stimulatory molecules on dendritic cells (DCs) among the splenocytes The splenocytes were stained for 30 min at 4°C in dark with the fluorochrome-conjugated anti-mouse antibodies: APC-anti-CD11c, Cy5.5-anti-CD86, and PE-anti-MHC II. After the staining process, the splenocytes were examined by flow cytometry. 2.13 Statistical analysis All the obtained data were expressed as mean ± standard deviation. Student’s t-test for independent means was conducted to distinguish the significant difference of the data (* meaning p

Cell Penetrating Peptides-based Redox-sensitive Vaccine Delivery

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