Immunomodulatory Nanoparticles from Elastin-Like Recombinamers

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Immunomodulatory Nanoparticles from Elastin-like Recombinamers: Single-Molecules for Tuberculosis Vaccine Development. Carmen García-Arévalo, Jesús F. Bermejo-Martín, Lucia Rico, Verónica Iglesias, Laura Martín, J. Carlos Rodríguez-Cabello, and F. Javier Arias Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300325v • Publication Date (Web): 09 Jan 2013 Downloaded from http://pubs.acs.org on January 18, 2013

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Molecular Pharmaceutics

Immunomodulatory

Nanoparticles

from Elastin-like Recombinamers: Single-

Molecules for Tuberculosis Vaccine Development

Carmen García-Arévaloa, Jesús F. Bermejo-Martínb, Lucia Ricob, Verónica Iglesiasb, Laura Martína, J. Carlos Rodríguez-Cabelloa, F. Javier Ariasa*.

a- Bioforge Group, University of Valladolid, CIBER-BBN, Paseo de Belén 11, 47011 Valladolid, b- Infection and Immunity Medical Research Unit (IMI), Microbiology Department, Hospital Clínico Universitario-IECSCYL, Ramón y Cajal 3, 47005 Valladolid, Spain

ABSTRACT This study investigates both the physicochemical properties and immunogenicity of a genetically engineered elastin-like block co-recombinamer (ELbcR) containing a major membrane protein sequence from Mycobacterium tuberculosis. The recombinant production of this ELbcR allows the production of large quantities of safe, antigenic particle-based constructs that directly and reversibly self-assemble into highly biocompatible, multivalent, monodisperse and stable nanovesicles with a diameter of 55 nm from the same gene product using a highly efficient and cost-effective inverse transition cycling (ITC) procedure. The compositional complexity of these vesicles is retained after secondary processes such as endotoxin removal, sterilization, and lyophilization. An initial

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pro-chemotactic cytokine response (IL-1β) followed by a pro-Th2/IL-5 response was observed in mice plasma following subcutaneous administration of the antigen-loaded nanovesicles in mice. This biphasic model of cytokine production was coupled with humoral isotype switching from IgM- to IgG-specific antibodies against the antigen, which was only observed in the presence of both the antigen and the polymer in the same construct and in the absence of additional adjuvants.

KEYWORDS:

Subunit vaccines, nanoparticles, elastin-like recombinamers (ELRs),

Mycobacterium tuberculosis INTRODUCTION The

engineering

of

materials

that

can

modulate

the

immune

system

(immunobioengineering) is an emerging field which, besides offering enormous potential for medical advances, is helping to provide new insights into basic immunobiology [1]. Biomaterials have become a key target of research for therapeutic purposes, such as vaccine development, as it has recently become possible to enhance vaccine efficacy by different approaches, including the ability to isolate and produce pure proteins and peptide antigens that are safer than more traditional vaccines [1-3]. Adjuvants have the ability to active antigen presenting cells (APCs), thereby resulting in the development of a long-lasting, antigen-specific immune response. Protein and/or peptide antigens are not usually sufficiently immunogenic to initiate dendritic cell (DC)-mediated adaptive immune responses in the absence of adjuvant factors [4]. As a result, the long-term efficacy of antigen vaccines depends on the development of effective adjuvants [4].


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Clearly, only adjuvants that induce minimal adverse effects will prove acceptable for standard prophylactic immunization in healthy individuals, although additional practical issues, including biodegradability, stability, ease of manufacture, cost, and applicability to a wide range of vaccines, are also important during their development [5]. Furthermore, the ideally formulated adjuvant must be chemically and physically well-defined in order to facilitate quality control and ensure reproducible manufacturing and activity [6]. Despite their relevance and essential roles, the list of clinically approved adjuvants is very limited. Indeed, aluminum hydroxide or phosphates (alum) were the only approved adjuvants in the USA for many years [7] until the US FDA approved the Cervarix® vaccine (GlaxoSmithKline Plc; Middlesex, UK), which contains both aluminum hydroxide and AS03/04 (3-O-desacyl-4'-monophosphoryl lipid A) as adjuvants, in late 2009. MF59, a sub-micron oil-in-water emulsion of a squalene, polyoxyethylene sorbitan monooleate (Tween 80) and sorbitan trioleate, was also approved in Europe and is currently found in several vaccines, such as an influenza vaccine manufactured by Novartis. These adjuvants primarily promote a Th2 or humoral response [7, 8]. One of the stumbling blocks in the development of anti-bacterial vaccines has been the lack of adjuvants that can effectively stimulate cell-mediated immunity whilst being safe enough for use in humans [7]. Biomaterials can be used to direct the nature of the immune response, as well as its magnitude, by varying factors such as particle size, surface properties, particle shape, and hydrophobicity, amongst others [1,9-10], as well as by including specific functional domains to promote receptor-specific endocytosis and increase specific cellular uptake [4], or programmed and controlled responses to changes in their environment or to external signals [11].



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Particle size is the primary control parameter when accessing sizes similar to, or even smaller than, those of biological viruses as it can be used to control the biological transport, and hence bioavailability, of a material to a remarkable degree. Additionally, the particles themselves are intrinsically recognized as a sign of danger [12]. As such, a particulate vaccine could possibly target any of these pathways, depending on its size and composition, including the incorporation of targeting ligands that are able to bind specific receptors [13]. In addition, hydrophilicity in a nano-formulation can modulate the amount of proteins adsorbed onto the surface of nanoparticles after administration, their opsonization, and can also increase their residence time in circulating blood [14]. The emerging field concerned with the construction of different self-assembled structures based on stimuli-responsive polymeric materials is becoming increasingly important in the development of new particle-based vaccines and adjuvants [15, 16], especially as some biomaterials, particularly polymers containing hydrophobic domains, can exhibit natural adjuvant behavior [4]. In this sense, the self-assembly of amphiphilic co-polymers that are able to undergo microphase separation into complex morphologies, with sequence-directed control of the material’s structure and properties, is also employed to form vesicles and micelles, within and upon which proteins can be incorporated [4, 17] The peptidic nature of protein-based block copolymers allows them to be both carefully designed at the genetic level and biosynthetically produced by recombinant synthesis. The advantages of using recombinant DNA approaches to produce these self-assembled structures include (i) the precise and specific structural properties that can be obtained, which yield the desired functional features of the polymer, such as hydrophobicity, secondary structures, and biorecognizable motifs, (ii) highly monodisperse molecular weights that are critical for


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producing distinct pharmacokinetic profiles, (iii) the absence of aggressive organic solvents during synthesis and purification, (iv) the fact that they provide biological functionality without potential infectivity, and (v) an ability to rapidly and systematically vary their chemical structures to produce libraries of polymers that differ in only specific amino acids, thus allowing the establishment of precise structure-function relationships. In addition, an increased structural complexity of the polymer does not result in a significant increase in production costs, thus meaning that their production is relatively inexpensive [18, 19, 20]. However, it is important to note that the quality control of recombinant materials varies substantially between research and medical grades, thus meaning that biocompatibility becomes an important issue when genetically engineered materials are used for biomedical applications [17]. Elastin-like recombinant polymers, a relatively new subclass of protein-based recombinant polymers, are composed of the pentapeptide repeat Val-Pro-Gly-Xaa-Gly (VPGXG), which mimics the hydrophobic domain of tropoelastin (X represents any natural or modified amino acid except proline) [22]. Recombinant forms of elastin-inspired polymers, recently renamed as “elastin-like recombinamers” (ELRs) [23], have allowed the formation of a wide range of biomaterial-based constructs and composites, such as aggregates [24], films [26, 27], fibers [27, 28], micelles [29, 30], nanoparticles [29, 31], and hydrogels [32], either alone or as hybrid systems, in a wide range of sizes, morphologies, and functional possibilities, all of which benefit from elastin’s elasticity and ability to self-assemble [25]. The lack of immunogenicity already described for ELRs, along with their biodegradability and biocompatibility for human tissue, tissue fluids, and blood, make these polymers exceptional candidates as carriers in vaccine-delivery approaches [33, 34]. Likewise, the



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modular nature of ELRs allows different amphiphilic elastin-like block co-recombinamers (ELbcRs), which are able to self-assemble with high reproducibility and homogeneity in a thermally driven process in an aqueous medium, to be constructed. In the light of previous work [24, 29, 35], the incorporation of antigenic molecule(s) at the hydrophilic terminus of an ELbcR by genetically encoded synthesis, without disrupting the assembly of the resulting nanoparticle, should theoretically allow presentation of the antigenic molecule(s) whilst the elastin support, which serves only as a vehicle, remains hidden from the immune system. Previous studies dealing with similar amphiphilic ELbcRs have stressed the numerous advantages of this kind of construct, which self-assembles into nanoparticles with the desired domains displayed at their surface, for drug-delivery and gene-therapy applications [35], but have seldom dealt with the development of subunit vaccines. One of the most relevant publications in this sense describes the expression and immunogenicity of a construct produced by combining plant-based production and the ELR fusion strategy to produce a potential vaccine candidate containing two major antigens from the bacterium M. tuberculosis (ESAT-6 and Ag85B) [36]. However, although the authors of that study verified that inclusion of the ELR increased the rate of antigen expression and accumulation in the plant, several issues associated with the purification limited the possibility of obtaining pure formulations, and therefore allowing the immunogenicity associated with the construct per se to be analyzed, without needing to incorporate additional adjuvants. ELbcRs could provide several of the desired characteristics of an ideal particle-based vaccine, including the possibility to develop a single gene product that assembles into


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nanoparticles displaying the desired antigenic sequence(s) by using a simple, low cost, reproducible and easily scalable bioproduction process without having to resort to bioconjugate chemistry techniques [22]. Furthermore, ELbcRs can be subjected to secondary processes such as sterilization, drying, packaging and reconstitution of the resulting dried powder [19, 37]. ELbcRs may therefore be a potential solution to the biological delivery challenges and weak immunogenicity of many antigens in order to target antigen presenting cells (APCs) in new vaccination strategies. In the present work, an immunodominant antigenic sequence from a major membrane constituent of M. tuberculosis (peptide sequence 91-110 from the 16 kDa antigen) [38] has been “adjuvanted” by recombinant linking to an amphiphilic ELbcR composed of a hydrophilic block based on glutamic acid (E) as guest residue at the amino terminus (E50) and a hydrophobic block based on isoleucine pentamers (I) at the carboxy terminus (I60). The 16 kDa antigen (Ag) contains murine and human T‐cell epitopes that induce T helper 1 (Th1) responses when assayed in vitro [38]. The physicochemical properties and immunogenicity of the resulting construct (Ag-E50I60) have been determined and compared with the individual constituents (Ag and E50I60) after ELbcR self-assembly.

MATERIALS AND METHODS ELbcR Design, Expression and Purification Standard molecular biology protocols were used during DNA manipulation and gene synthesis. Sequential introduction of the repetitive ELR polypeptide-coding gene segments



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to form fusion genes with a fully controlled composition and chain length was carried out using a “recursive directional ligation” (RDL) strategy [39]. The sequences were verified by agarose gel electrophoresis of the restriction fragments generated after enzymatic digestion and automated DNA sequencing. Selected genes were subcloned into a modified pET-25(+) expression vector then transformed into the E. coli strain BLR(DE3). All expression conditions and purification protocols have been described previously [37] and consisted of three sequential rounds of inverse transition cycling, followed by a secondary treatment for endotoxin removal [41] (Supplementary information). The purity and molecular weight of the co-recombinamers was routinely determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MALDI-TOF/MS). The amino acid composition was further verified by HPLC analysis [24] (Supplementary information).

Sample Preparation Buffered aqueous solutions of the ELbcRs were prepared from lyophilized purified specimens dissolved at the appropriate concentration in cold phosphate buffered saline (PBS) at pH 7.4, then filtered using a 0.45 µm PVDF syringe filter.

Turbidity and Differential Scanning Calorimetry (DSC)


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Turbidity experiments were conducted using a Varian Cary 50 UV-Vis spectrophotometer (Varian Inc., NC, USA) equipped with a thermostatted sample chamber. Optical density (OD) was assessed by the change in absorbance of a 25 µM ELbcR solution in phosphate buffered saline (PBS), at 350 nm, as a function of temperature in the range 5.0–60.0 °C (ramp 0.2 °C/min). Samples were stabilized at each temperature until a constant turbidity value was reached. Three independent readings were taken at each temperature. DSC experiments were performed using a Mettler Toledo 822e apparatus equipped with a liquid-nitrogen cooler. Both temperature and enthalpy were calibrated against an indium standard. A 20 µL aliquot of each solution at 50 mg/mL in PBS (pH 7.4), and its corresponding PBS reference, were subjected to an initial isothermal stage (5 min at 0 °C to stabilize the temperature and state of the samples), followed by heating at 5 °C/min from 0 °C to 40 °C. A further 4.5 consecutive DSC heating-cooling cycles were performed at 5 °C/min from 0 to 40 °C and from 40 °C to 0 °C, after 5 min at 0 °C.

Dynamic (DLS) and Static Light Scattering (SLS) The refractive index (nD) was measured for PBS solutions of the ELbcRs over a wide concentration range (0.2–3.40×10-3 g/mL) at a fixed temperature of 37 °C using a digital refractometer (Mettler Toledo RE50, Mettler-Toledo GmbH, Schwerzenbach, Switzerland) equipped with a Peltier thermostat. Light scattering measurements were performed as described previously [23] using a BI200SM multiangle goniometer (Brookhaven Instrument Corp., NY, USA) equipped with a



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33mW He-Ne vertically polarized laser (wavelength: 632.8 nm) and a digital correlator (BI-9000AT). DLS measurements were performed at a scattering angle of 90° using cold-filtered ELbcR solutions at a concentration of 20 µM in PBS to calculate the size distribution (hydrodynamic radius; Rh) and polydispersity index (PDI) at different temperatures in the range 8–50 °C from three different samples. The long-term size stability of ELbcR solutions at 37 °C was evaluated up to one year in PBS and in 0.1% PBS-BSA (pH 7.4). SLS measurements were performed using cold-filtered ELbcR solutions in PBS equilibrated at 37 °C at concentrations in the range 0.5–5.0 mg/mL and scattering angles of between 50° and 130°. The resulting data were fitted and used to determine the gyration radius (Rg) and molecular weight of the aggregates (Mw,agg).

Zeta Potential The particle-velocity distribution (electrophoretic-mobility) was determined by Laser Doppler Velocimetry, and the zeta potential subsequently calculated, using freshly prepared and filtered samples dissolved in both water (pH 7.4) and PBS using a Zetasizer Nano Series (Malvern, UK). The samples were analyzed at 37 °C by performing 10 readings.

Surface Tension by Pendant Drop Technique


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The critical aggregation concentration (CAC) of the different ELbcR solutions in PBS was determined from surface tension measurements derived from a drop-shape analysis using the pendant drop technique [42]. The changes in the shape of the resulting drop at the air/water interface upon increasing the ELbcR concentration previously stabilized at 37 °C for 5 min from a blank solution of PBS to 20 µM ELbcR were monitored using the SCA 20 software of a Data Physics OCA20 instrument, which outlines and scales the profile of the drop hanging from a straight precision dosing needle. The drops (4 µL at 0.5 µL/s) were infused using a 500 µL Gastight® Hamilton syringe. At least three drops per condition were analyzed and the CAC determined from the point of slope change after plotting the change in surface tension values versus log(concentration) of the ELbcRs.

Transmission Electron Microscopy (TEM) Buffered solutions of the ELbcRs were prepared by placing a drop of the stabilized solution (20 µM in PBS; pH 7.4), heated at 37 °C for at least 15 minutes and stained with uranyl acetate solution (1.0 wt %), on a carbon-coated copper grid, followed by solvent evaporation. Further samples were also prepared for cryo-TEM by rapid vitrification of liquid samples using an automated Vitrobot™ Mark IV (FEI) vitrification robot. Specimens were subsequently observed using a JEM-2200FS/CR transmission electron microscope (JEOL, Japan), equipped with an ULTRASCAN 4000 SP (4008×4008 pixels) cooled slow-scan CCD camera (GATAN, UK).

Atomic Force Microscopy (AFM)



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AFM samples were prepared by placing 50 µL of the ELbcR at a concentration of 20 µM stabilized at 37 °C in PBS on mica, followed by water evaporation at the same temperature. AFM was performed in air at 25 °C using a Multimode 8 AFM attached to a Nanoscope V electronics apparatus (Veeco Instruments, Santa Barbara, CA) in tapping mode. Both topography and phase-signal images were recorded with a resolution of 512 x 512 data points.

Preparation of Vaccine Doses Five groups of vaccine doses were prepared for immunization. Two groups were immunized with 50 and 100 µg of Ag-E50I60, respectively. Control groups consisted of E50I60 and the purified 20-mer antigen (Ag) (Biomatik Corporation, Ontario, Canada) administered at the same molar concentration as the maximum dose of Ag-E50I60 (95.5 and 3.94 µg, respectively). A third control was treated with the buffered solvent vehicle (PBS) only. Prior to administration, all doses were filtered and kept in a bath at 37 °C for 30 minutes.

In vivo immunization and sampling in a mouse model A total of 40 BALB/cByJ female mice aged between 6 and 10 weeks, supplied by Charles River Laboratories, Lyon (France), were randomly distributed into five groups and immunized as described previously. Immunization consisted of subcutaneous injections (200 µL in PBS) on days 0, 14, and 28. All experiments were conducted in accordance with national guidelines for animal care. Weight loss, water intake and clinical conditions (neurological status, presence of bleeding, activity) were monitored throughout the


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experiment. At the end of the experiment, mice were humanely euthanized by cervical dislocation. Blood samples (0.5 mL) were taken from the tail on days 0, 1, 15 and 29 for cytokine determination, and on days 7, 21 and 35 for immunoglobulin quantification, using a Microvette® CB 300 K2E (SARSTEDT, Toronto, Canada). In all cases blood was allowed to set at room temperature and then centrifuged for 10 minutes at 2500 rpm. Plasma samples were collected individually into Eppendorf tubes and maintained at –20 °C in the freezer until use so that all parameters could be determined in a single experiment

Cytokine Profiling and Antibody Response Cytokine levels (day 1, 15 and 29) were determined using the multiplex Biorad© 8 plex immunoassay (Hercules, CA, USA). Detection limits (pg/mL) were: IL-1β (2.46), IL-2 (3.89), IL-4 (6.78), IL-5 (2.76), IL-10 (1.3), GM-CSF (2.13), IFN-γ (3.35), TNF-α (3.46). All samples below that were considered as equal to the detection limit. IgG- and IgM-specific immunoglobulin loads were determined by indirect ELISA on days 7, 21 and 35. Four different ELISAs were mounted by coating plates with Ag-E50I60 (200 ng/well), E50I60 (200 ng/well), biotinylated Ag (100 ng/well; Biomatik Corporation, Ontario, Canada) and a culture filtrate protein (CFP) from a diagnosed M. tuberculosis clinical isolate (500 ng/well) kindly provided by Dr. O. M. Rivero Lezcano from the complex care research unit at the Hospital de Leon (Spain). Plasma samples (1/100 dilution) were analyzed in triplicate. Immunoglobulin levels were determined using horseradish peroxidase-labeled monoclonal antibodies against mouse immunoglobulins



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IgM (1:25,000 dilution, µ-chain specific peroxidase conjugate. Sigma St. Quentin, France) and IgG (1:10,000 dilution, γ-chain specific peroxidase conjugate. Sigma St. Quentin, France). Absorbance was measured at 370 nm after revelation with supersensitive 3,3ʹ,5,5ʹ-tetramethylbenzidine liquid substrate (TMB, Sigma, St. Quentin, France).

Statistical analysis Cytokine levels were compared by using the Mann–Whitney U test for unrelated groups and the Wilcoxon signed rank test for paired samples. All statistical tests were two-sided; p

Immunomodulatory Nanoparticles from Elastin-Like Recombinamers

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