Enhancing the Magnitude of Antibody Responses through Biomaterial

Jun 5, 2015 - Controls included cultures fixed with absolute ethanol or cells treated with media containing no peptide. All groups contained three ...
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Enhancing the Magnitude of Antibody Responses through Biomaterial Stereochemistry Rajagopal Appavu,† Charles B. Chesson,‡,§ Alexey Y. Koyfman,† Joshua D. Snook,† Frederick J. Kohlhapp,∥ Andrew Zloza,∥ and Jai S. Rudra*,†,§ †

Department of Pharmacology & Toxicology, ‡Institute for Translational Sciences, and §Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas 77555, United States ∥ Departments of Microbiology/Immunology and Internal Medicine, Rush University Medical Center, Chicago, Illinois 60612, United States ABSTRACT: D-Amino acid analogs of peptides and proteins are attractive for applications in biotechnology and medicine due to their reduced proteolytic sensitivity. Here, we report that self-assembling peptide nanofibers composed of D-amino acids act as immune adjuvants, and investigate their ability to induce antibody responses in comparison to their L-amino acid counterparts. The model antigenic peptide OVA (chicken egg ovalbumin aa 323−339) from chicken egg ovalbumin, known to elicit antibody responses in mice, was linked to an L- or Damino acid self-assembling peptide domain to generate enantiomeric nanofibers displaying the same epitope. The chiral nature of the fusion peptides was confirmed by circular dichrosim spectroscopy and transmission electron microscopy studies indicated that OVA-bearing enantiomers self-assembled into nanofibers with similar morphologies. In mice, D-amino acid peptide nanofibers displaying OVA elicited stronger antibody responses, equivalent levels of CD4+ T cell responses, and long-term antigen-presentation in vivo compared to L-amino acid nanofibers. Our findings indicate that self-assembling peptides composed of D-amino acids are strong immune adjuvants and that biomaterial stereochemistry can be used as a design tool to program adaptive immune responses for vaccine development. KEYWORDS: adjuvant, enantiomer, nanofiber, peptide, stereochemistry, self-assembly

1. INTRODUCTION Biomaterials constructed from self-assembling peptides have attracted considerable interest for applications in biotechnology and medicine because of their ease of synthesis, biocompatibility, and biodegradation properties.1−7 Recently, self-assembling peptides that form β-sheet rich nanofibers have been reported to act as effective immune adjuvants and elicit strong antibody and cellular immune responses in mice.8−12 When a peptide or protein antigen is covalently linked to a selfassembling peptide domain the resulting nanofibers display the antigen on their surface in a multivalent fashion.8 Immunization with antigen-bearing peptide nanofibers has been shown to be protective in murine models of malaria,10 cancer,11 and influenza.12 Moreover, this phenomenon was not limited to a single self-assembling peptide domain, route of administration, disease model, or mouse strain.9,11 Also, no immune responses have been detected against self-assembling peptides themselves even when administered with strong exogenous adjuvants.8 This is of immense interest for applications in vaccine development and immunotherapy because of the toxicity and compositional heterogeneity of currently used immune adjuvants.13 Because naturally occurring proteins utilize only L-form amino acids, most self-assembling peptides designed to date have utilized naturally occurring L-amino acids.14 Although D© XXXX American Chemical Society

amino acids very rarely participate in protein synthesis, they are vital to all living organisms including bacteria (D-alanine is a component of the cell wall) and mammals (D-serine is involved in glutamatergic neurotransmission in the central nervous system).15,16 In humans, physiological fluids such as plasma, cerebrospinal fluid, and amniotic fluid have been reported to contain high levels of D-amino acids.17 In an effort to control the rate of protease-mediated degradation and enhance stability of peptide therapeutics in vivo, one logical strategy that has been used is the full or partial replacement of L-amino acids with their D-enantiomers. D-Amino acid peptides are resistant to proteolysis and have numerous applications in microbiology, physiology, and medicine.18−20 In the course of applying this strategy to self-assembling peptides, Zhang and co-workers first reported the effects of stereochemistry on the assembly and behavior of selfassembling peptide biomaterials using the enantiomers LEAK16 and D-EAK16.14,21 Although both peptides assembled into well-ordered nanofibers with mirror image secondary structures, significant differences were observed in responses to external stimuli like pH, temperature, and the presence of Received: March 19, 2015 Accepted: June 5, 2015

A

DOI: 10.1021/acsbiomaterials.5b00139 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Table 1. List of Peptides Investigated in This Study, with Abbreviations, Peptide Sequences, Theoretical Mass, and Observed Mass peptides

sequences

calcd mass

obsd [M + H]+

L

Ac-FKFEFKFE-Am Ac-DFDKDFDEDFDKDFDE-Am H2N-ISQAVHAAHAEINEAGR−OH H2N-ISQAVHAAHAEINEAGRSGSGFKFEFKFE-Am H2N-ISQAVHAAHAEINEAGRSGSGDFDKDFDEDFDKDFDE-Am

1161.57 1161.57 1773.90 3164.46 3164.46

1162.02 1162.60 1774.77 3164.58 3165.02

KFE8 KFE8D OVA OVA-KFE8L OVA-KFE8D

denaturing agents or proteases.21 Using FRET studies and the model amphipathic self-assembling peptide KFE8, Nilsson and co-workers demonstrated that equimolar mixtures of selfassembling peptide enantiomers pack into “rippled β-sheet” nanofibers composed of alternating L- and D-peptides.22 This alternate packing had an enthalpic advantage over all-L or all-D nanofiber assemblies, which was confirmed by Schneider and Pochan, who observed that racemic hydrogels of the β-hairpin peptide MAX1 and its enantiomer DMAX1 exhibited maximum rigidity compared to the individual peptides or any other ratio of the enantiomers.23 Luo et al. have reported that D-amino acid self-assembling peptide hydrogels were effective at supporting in vitro cell cultures, homeostasis and wound healing in animal models, and resisting proteolytic degradation making them attractive for applications in tissue engineering, regenerative medicine, and drug delivery.24 Because of their chiral nature and inherent reduced susceptibility to proteolysis, D-amino acid self-assembling peptides are attractive biomaterial platforms for vaccine development and immunotherapy. In this study, we investigated the effect of stereochemistry of the self-assembling peptide domain on antibody responses using the wellcharacterized amphipathic self-assembling peptide domain KFE8 (FKFEFKFE) and the model peptide antigen OVA (ISQAVHAAHAEINEAGR, aa 323−339 chicken egg ovalbumin). OVA is a MHC-Class II (I-Ab)-restricted epitope that is recognized by both CD4+ T cells and B cells and induces CD4+ T cell dependent antibody responses in mice when linked to self-assembling peptides.8,9 Here, OVA was linked to the Nterminus of the self-assembling motif KFE8 containing all L(KFE8L) or all D-(KFE8D) amino acids using a short amino acid linker (SGSG). The OVA-bearing enantiomeric peptides differ only in the chirality of the self-assembling domain, which allows for direct comparison of the effect of stereochemistry on antibody responses. Mice were vaccinated with OVA-bearing KFE8 enantiomers and the magnitude and nature of antibody responses was evaluated along with CD4+ T helper cell responses. We also investigated persistence of the D-form nanofibers at the immunization site. Our results indicate that Damino acid peptide nanofibers enhanced the magnitude of the antibody response and persisted longer at the immunization site compared to their enantiomeric counterparts. This suggests that stereochemistry can be used as a design tool to modulate immunological properties of self-assembling peptide biomaterials for applications in vaccine development and immunotherapies.

peptides were double coupled using HBTU (O-(benzotriazol1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) and HOBt (1-Hydroxybenzotriazole) chemistries. Peptides were cleaved from the resin using a 95% TFA/2.5% water/ 2.5% triisopropyl silane cocktail and washed in diethyl ether. The crude product was purified by reverse-phase HPLC (C18 column) using acetonitrile/H2O gradients of >90% purity and peptide mass was confirmed by MALDI using α-cyano-4hydroxycinnamic acid matrix (Bruker Daltonics, MA). All peptides were lyophilized and stored at 4 °C. Endotoxin levels of all peptides were tested using a limulus amebocyte lystae (LAL) chromogenic end point assay (Lonza, MA) at the same volume and peptide concentration used for immunizations and were found to be less than 0.11 EU/mL and within acceptable limits.25 To account for batch-to-batch variability, we synthesized three different sets of peptides for immunizing mice. 2.2. Transmission Electron Microscopy (TEM). Stock solutions of 1 mM peptides were allowed to fibrillize in water overnight at room temperature, diluted in PBS to 0.3 mM, and applied to 300 mesh copper grids with carbon support film (Quantifoil). The grids were negatively stained with 2% uranyl acetate, and imaged on a JEM1400 TEM (JEOL) equipped with LaB6 electron gun and digital cameras. Images were viewed and recorded with an Ultrascan 1000 camera (Gatan). 2.3. Circular Dichroism Spectroscopy (CD). CD-experiments were carried out on a JASCO J-815 CD-Spectrometer. Peptide stock solutions (1 mM) were made in ultra pure water and diluted to working concentrations before use. The CD-wavelength range was from 260 to 195 nm with a scanning speed of 0.3 nm/s and a bandwidth of 0.5 nm. CD-spectra were recorded at room temperature with a fixed-path-length (1 mm) cell. The solvent background contribution was subtracted and resultant CD-signal was converted to mean residue ellipticity. 2.4. Cytotoxicity Assay. A standard MTS assay was utilized to determine peptide cytotoxicity (Promega, WI). Mouse lymphocytes (B6 mice, 100,000 cells/well) were seeded in 96-well plates in culture media (1640 rpmI containing 1% penicillin/streptomycin, 2% Lglutamine, and 10% FBS) containing 0.01, 0.1, or 1 mg/mL of KFE8L or KFE8D peptide. The cells were incubated for 24 h and the medium was replaced. MTS reagent was applied for 4 h and absorbance at 490 nm was measured using a microplate reader (Biotek, NH). Controls included cultures fixed with absolute ethanol or cells treated with media containing no peptide. All groups contained three replicates. 2.5. In Vitro Activation Assay. To assess the processing and presentation of OVA-KFE8L and OVA-KFE8D nanofibers, we utilized a transgenic mouse model whose CD4+ T cell receptors recognize the OVA323−339 epitope in the context of MHC class II molecules. OT-II transgenic mice (B6.Cg-Tg[TcraTcrb]425Cbn/J) mice were purchased from Jackson laboratories at 7−8 weeks of age. The OVAbearing enantiomers were coated onto 96-well plates (20 μg/mL, 100 μL/well) and incubated at 4 °C for 24 h. Whole lymphocyte extracts (containing antigen-presenting cells, B cells, and CD4+ T cell populations) from inguinal, axial, and brachial lymph nodes of OT-II mice were extracted and washed twice with complete RPMI (cRPMI) media. This extract was then added to wells coated with OVA-bearing enantiomers and 6 h later the production of cytokines IFN-γ and IL-2

2. MATERIALS AND METHODS 2.1. Peptide Synthesis and Purification. All peptides (sequences in Table 1) were synthesized using standard Fmoc

Chemistry on a CS Bio-CS336X solid phase peptide synthesizer (CS Bio, CA). Rink Amide MBHA or Wang resin (Novabiochem, MA) was swelled in dry DMF for 1 h, and B

DOI: 10.1021/acsbiomaterials.5b00139 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Structures of self-assembling peptide enantiomers (A) KFE8L and (B) KFE8D. TEM micrographs showing nanofibers of (C) KFE8L and (D) KFE8D. The scale bar shown is 50 nm. (E) Circular dichroism spectra of the enantiomers in water at 0.25 mM indicating beta-sheet rich structures and the spectra are mirror images reflecting molecular chirality. (F) MTS assay data showing noncytotoxicity of KFE8D nanofibers in primary cell cultures compared to KFE8D nanofibers. Cultures fixed with absolute ethanol or culture media without added peptides are shown as controls. *p < 0.05 by ANOVA using Tukey posthoc test. Scale bar = 200 nm. were measured using flow cytometry. Golgiplug (BD-Biosciences, NJ) was added to a final dilution of 1:1000 and 600 000 cells were added to wells coated with the enantiomers. Cells cultured on PBS-coated wells or with free OVA peptide (1 μg/mL) added in the culture media were used as negative and positive controls, respectively. The plates were spun at 300 rpm for 2 min prior to being incubated at 37 °C for 6 h. The cells were then washed twice in PBS and analyzed using flow cytometry. 2.6. Flow Cytometry. Cells were stained for surface markers CD4 (APC-Cy7, Clone GK1.5), CD3 (Pacific Blue, Clone 145−2C11) and live/dead stain (eBioscience, CA) for 30 min at 4 °C. Cells were washed twice with PBS, permeablized, and stained for intracellular cytokines IFN-γ (PE-Cy7, Clone XMG1.2), and IL-2 (APC, Clone JES6−5H4) (eBioscience, CA) and analyzed using flow cytometry on a BD-Biosciences LSR-II and analyzed with FlowJo software. Cells were gated on live, singlet CD4+ lymphocytes as follows, FSC-A v SSC-A → SSC-A v Live/DEAD-→ FSA-A v FSC-H → SSC-A v SSCH → FSC-W v SSC-W → CD3+/CD4+. Fluorophore overlap was compensated by single positive control staining for murine CD4 with each fluorophore utilized. All experiments were performed in triplicate and repeated twice. 2.7. Animals and Immunizations. Female mice (C57BL/6, 6−8 weeks old) were purchased from Taconic Farms. For vaccination, the stock solutions of peptide nanofibers were prepared freshly each time. First the peptides were dissolved in water (8 mM stock), incubated overnight, and diluted 4-fold in PBS (2 mM stock) 4 h prior to allow fibrillization. At the injected concentration, the nanofiber solution was viscous gel and could be easily manipulated using a 25-gauge syringe. To investigate antibody responses and ensure all groups received equal amounts of antigen, 50 μL of OVA-bearing enantiomers (50 μL of 2 mM stock in PBS corresponding to 100 nm of antigen) were injected subcutaneously in the flank at two different sites. Mice were boosted on day 28 with two 25 μL injections of the enantiomers or controls (50 nmol of antigen) and sacrificed on day 42. Blood was collected weekly via the submandibular vein and sera stored at −80 °C. Mice immunized with free OVA peptide in PBS or Montanide ISA 720 (ISA 720) adjuvant were used as controls. ISA 720 is a vaccine adjuvant composed of metabolizable oil and a surfactant system designed to make a water-in-oil emulsion. It is nontoxic and biodegradable and has been used in more than 50 clinical trials related to therapeutic vaccines against various diseases and has been shown to be a better alternative to Freund’s adjuvant in clinical trials.26 ISA-720 emulsion was prepared

by mixing equal volumes of peptide solution and adjuvant immediately prior to immunization. All experiments were conducted under approved protocols by the University of Texas Medical Branch Institutional Animal Care and Use Committee and repeated independently three times with 3−5 mice per group per experiment. 2.8. Antibody Responses. High-binding ELISA plates (eBioscience, CA) were coated with 20 μg/mL of antigen in PBS overnight at 4 °C and blocked with 200 μL of 1% BSA in PBST (0.5% Tween-20 in PBS) for 1 h. Serum dilutions were applied (1:1 × 10−2 to 1:1 × 10−9, 100 μL/well) for 1 h at room temperature followed by peroxidase-conjugated goat antimouse IgG (H+L) (Jackson Immuno Research, PA) (1:5000 in 1% BSA-PBST, 100 μL/well). Plates were developed using TMB substrate (100 μL/well, eBioscience, CA), the reaction stopped using 50 μL of 1 M phosphoric acid, and absorbance measured at 450 nm. Absorbance values of PBS (no antigen)-coated wells were subtracted to account for background. Antibody isotypes were determined using a mouse monoclonal antibody kit (Sigma, MO) with secondary goat antimouse IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA. 2.9. Surface Plasmon Resonance. Antibody levels in mice immunized with OVA-bearing enantiomers or controls were also analyzed using surface plasmon resonance on a Biacore T100 (GE Healthcare) system. Biotin-OVA (50 ng/mL) peptide was dissolved in HBS-EP+ buffer (GE Healthcare) and immobilized on streptavidincoated chip (BR-1005−31, GE Healthcare). Serum from mice was pooled and diluted in HBS-EP+ buffer (1:2000) prior to flowing. Pooled sera (300 μL) were flown over the biotin-OVA functionalized chip for 600 s, followed by a 600 s wait. The chip was regenerated using 1 M NaCl for 1000 s between replicates and regenerated twice between different groups. All experiments were performed at room temperature and repeated in triplicate. 2.10. In Vivo Antigen-Presentation Assay. To assess the persistence of OVA-KFE8D nanofibers at the immunization site, we utilized the transgenic OT-II mouse model whose T cells recognize the OVA epitope in the context of MHC-class II molecules. Splenocytes from OT-II mice were isolated into single cell suspensions and purified using negative selection for CD3+ CD4+ cells according to the manufacturers recommended protocol (STEMcell technologies, Vancouver, BC). The purified CD4+ T cells were then labeled with 10 μM carboxyfluorescein (CFSE) for 10 min. Cells were then washed twice with PBS and resuspended in 400 μL of cRPMI media. Mice were immunized in the footpad with OVA-bearing enantiomers (20 C

DOI: 10.1021/acsbiomaterials.5b00139 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering μL of 2 mM solution) and 7 days later, 500 000 CFSE-labeled OT-II cells were adoptively transferred into the footpad of each mouse. After 24 h, the mice were sacrificed and the draining lymph nodes (inguinal and popliteal) were excised. CFSE dilution in response to the presence of antigen was analyzed using flow cytometry on a BD-Biosciences LSR-II and analyzed with FlowJo software. Spectral compensation and gating on live, singlet, nondebris CD4+ T lymphocytes was performed as described above. 2.11. Statistical Analysis. All the experimental data were plotted using GraphPad Prism software and represented as mean ± SEM, and statistical analysis was performed by ANOVA with Tukey’s post hoc test. Statistical significance was assigned at p values