The red alga Porphyridium as a host for molecular farming: Efficient production of immunologically active hepatitis C virus glycoprotein.

Microalgae are promising production platforms for the cost-effective production of recombinant proteins. We have recently established that the red alga Porphyridium purpureum provides superior transgene expression properties, due to the episomal maintenance of transformation vectors as multicopy plasmids in the nucleus. Here, we have explored the potential of Porphyridium to synthesize complex pharmaceutical proteins to high levels. Testing expression constructs for a candidate subunit vaccine against the hepatitis C virus (HCV), we show that the soluble HCV E2 glycoprotein can be produced in transgenic algal cultures to high levels. The antigen undergoes faithful posttranslational modification by N-glycosylation and is recognized by conformationally selective antibodies, suggesting that it adopts a proper antigenic conformation in the endoplasmic reticulum of red algal cells. We also report the experimental determination of the structure of the N-glycan moiety that is attached to glycosylated proteins in Porphyridium. Finally, we demonstrate the immunogenicity of the HCV antigen produced in red algae when administered by injection as pure protein or by feeding of algal biomass.

Generation of a DSF-Guided Refolded Bacterially Expressed Hemagglutinin Ectodomain of Influenza Virus A/Puerto Rico/8/1934 H1N1 as a Model for Influenza Vaccine Antigens.

Influenza virus infections represent an ongoing public health threat as well as an economic burden. Although seasonal influenza vaccines have been available for some decades, efforts are being made to generate new efficient, flexible, and cost-effective technologies to be transferred into production. Our work describes the development of a model influenza hemagglutinin antigen that is capable of inducing protection against viral challenge in mice. High amounts of the H1 hemagglutinin ectodomain, HA18-528, were expressed in a bacterial system as insoluble inclusion bodies. Solubilization was followed by a thorough differential scanning fluorimetry (DSF)-guided optimization of refolding, which allows for fast and reliable screening of several refolding conditions, yielding tens of milligrams/L of folded protein. Structural and functional analysis revealed native-like folding as well as the presence of a mix of monomers and oligomers in solution. Mice immunized with HA18-528 were protected when exposed to influenza A virus as opposed to mice that received full-length denatured protein. Sera of mice immunized with HA18-528 showed both high titers of antigen-specific IgG1 and IgG2a isotypes as well as viral neutralization activity. These results prove the feasibility of the recombinant bacterial expression system coupled with DSF-guided refolding in providing influenza hemagglutinin for vaccine development.

Fever as an evolutionary agent to select immune complexes interfaces.

We herein analyzed all available protein-protein interfaces of the immune complexes from the Protein Data Bank whose antigens belong to pathogens or cancers that are modulated by fever in mammalian hosts. We also included, for comparison, protein interfaces from immune complexes that are not significantly modulated by the fever response. We highlight the distribution of amino acids at these viral, bacterial, protozoan and cancer epitopes, and at their corresponding paratopes that belong strictly to monoclonal antibodies. We identify the "hotspots", i.e. residues that are highly connected at such interfaces, and assess the structural, kinetic and thermodynamic parameters responsible for complex formation. We argue for an evolutionary pressure for the types of residues at these protein interfaces that may explain the role of fever as a selective force for optimizing antibody binding to antigens.

Nickel (II) and Cobalt (II) Alginate Biopolymers as a "Carry and Release" Platform for Polyhistidine-Tagged Proteins.

Protein immobilization using biopolymer scaffolds generally involves undesired protein loss of function due to denaturation, steric hindrance or improper orientation. Moreover, most methods for protein immobilization require expensive reagents and laborious procedures. This work presents the synthesis and proof of concept application of two alginate hydrogels that are able to bind proteins with polyhistidine tags by means of interaction with the crosslinking cations. Nickel (II) and cobalt (II) alginate hydrogels were prepared using a simple ionic gelation method. Hydrogels were characterized by optical microscopy and AFM, and evaluated for potential cytotoxicity. In addition, binding capacity was tested towards proteins with or without HisTAG. Hydrogels had moderate cytotoxicity and were able to exclusively bind polyhistidine-tagged proteins with a binding capacity of approximately 300 µg EGFP (enhanced green fluorescent protein) per 1 mL of hydrogel. A lyophilized hydrogel-protein complex dissolved upon the addition of PBS and allowed the protein release and regain of biological activity. In conclusion, the nickel (II) and cobalt (II) alginate biopolymers provided an excellent platform for the "carry and release" of polyhistidine-tagged proteins.

Low Molecular Weight and Polymeric Modifiers as Toughening Agents in Poly(3-Hydroxybutyrate) Films.

The inherent brittleness of poly(3-hydroxybutyrate) (PHB) prevents its use as a substitute of petroleum-based polymers. Low molecular weight plasticizers, such as tributyl 2-acetyl citrate (TAC), cannot properly solve this issue. Herein, PHB films were obtained using a biosynthesized poly(3-hydroxyoctanoate) (PHO) and a commercially available TAC as toughening agents. The use of TAC strongly decreased the PHB thermal stability up to 200 °C due to the loss of low boiling point plasticizer, while minor weight loss was noticed at this temperature for the PHB-PHO blend. Both agents shifted the glass transition temperature of PHB to a lower temperature, the effect being more pronounced for TAC. The elongation at break of PHB increased by 700% after PHO addition and by only 185% in the case of TAC; this demonstrates an important toughening effect of the polymeric modifier. Migration of TAC to the upper surface of the films and no sign of migration in the case of PHO were highlighted by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) results. In vitro biocompatibility tests showed that all the PHB films are non-toxic towards L929 cells and have no proinflammatory immune response. The use of PHO as a toughening agent in PHB represents an attractive solution to its brittleness in the case of packaging and biomedical applications while conserving its biodegradability and biocompatibility.

Composite Nanogels Based on Zeolite-Poly(ethylene glycol) Diacrylate for Controlled Drug Delivery.

This study presents the design of novel composites nanogels, based on poly(ethylene glycol) diacrylate and natural zeolite particles, that are able to act as materials with controlled drug delivery properties. Natural zeolite‒nanogels composite, with varying zeolite contents, were obtained by an inverse mini-emulsion technique and loaded with 5-fluorouracil, a widely used chemotherapeutic drug. Herein, the possibility of adjusting final properties by means of modifying the preparation conditions was investigated. The prepared composite nanogels are characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA). In light of this tunable drug-loading capability, swelling behaviour, and cytotoxicity, these composite nanogels could be highly attractive as drug reservoirs.

Comprehensive characterization of silica-modified silicon rubbers.

In this study a commercially liquid silicone rubber was filled with fumed silica particles in different concentrations and evaluated for medical applications. The thermal, morphological and mechanical properties of silicone/silica composite samples were studied before and after aging, flexural tests and immersion in saline environment. Understanding the effect of silica content, aging conditions and thickness (from 0.6 to 2 mm) of the samples on the behavior of these materials in different environments is crucial for applications as implantable devices. Before inducing any mechanical stress, tensile strength was found to increase for samples containing 3 or 5 wt% of fumed silica, depending on the thickness. A similar trend was observed after 106 flexes for tensile strength, storage modulus and hardness at room temperature, which increased with the concentration of fumed silica. Moreover, tensile strength decreased with increasing the thickness of the samples from 0.6 to 2 mm. The thermal degradation was found to start at higher temperature in the case of the composites as compared with neat silicone, however, the glass transition and melting temperatures were only slightly modified by the presence of the silica particles, regardless the mechanical aging. The MTT assay using L929 fibroblasts mouse cells showed a good short-time cytocompatibility for both silicone elastomer and the composite with 3 wt% fumed silica. Similarly, the measurement of the cytokine secretion revealed no inflammatory response.

Oral administration of a chimeric Hepatitis B Virus S/preS1 antigen produced in lettuce triggers infection neutralizing antibodies in mice.

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S) envelope protein are currently used in universal vaccination programs and achieve protective immune response in more than 90% of recipients. However, new vaccination strategies are necessary for successful immunization of the remaining non- or low-responders. We have previously characterized a novel HBV chimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L) envelope protein (genotype D). The S/preS121-47 chimera produced in mammalian cells and Nicotiana benthamiana plants, induced a significantly stronger immune response in parenterally vaccinated mice than the S protein. Here we describe the transient expression of the S/preS121-47 antigen in an edible plant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral administration of adjuvant-free Lactuca sativa expressing the S/preS121-47 antigen, three times, at 1 µg/dose, was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies were able to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) more efficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results support the S/preS121-47 antigen as a promising candidate for future development as an edible HBV vaccine.

Nanocomposite particles with improved microstructure for 3D culture systems and bone regeneration.

Nano-apatite and gelatin-alginate hydrogel microparticles have been prepared by a one-step synthesis combined with electrostatic bead generation, for the reconstruction of bone defects. Based on the analysis of bone composition, architecture and embryonic intramembranous ossification, a bio-inspired fabrication has been developed. Accordingly, the mineral phase has been in situ synthesized, calcifying the hydrogel matrix while the latter was crosslinked, finally generating microparticles that can assemble into a bone defect to ensure interconnected pores. Although nano-apatite-biopolymer composites have been widely investigated, microstructural optimization to provide improved distribution and stability of the mineral is rarely achieved. The optimization of the developed method progressively resulted in two types of formulations (15P and 7.5P), with 15 and 7.5 (wt%) phosphate content in the initial precursor. The osteolytic potential was investigated using differentiated macrophages. A commercially available calcium phosphate bone graft substitute (Eurocer 400) was incorporated into the hydrogel, and the obtained composites were in vitro tested for comparison. The cytocompatibility of the microparticles was studied with mouse osteoblast-like cell line MC3T3-E1. Results indicated the best in vitro performance have been obtained for the sample loaded with 7.5P. Preliminary evaluation of biocompatibility into a critical size (3 mm) defect in rabbits showed that 7.5P nanocomposite is associated with newly formed bone in the proximity of the microparticles, after 28 days.

Medium Chain-Length Polyhydroxyalkanoate Copolymer Modified by Bacterial Cellulose for Medical Devices.

Medium chain-length polyhydroxyalkanoates (mPHAs) are flexible elastomeric biopolymers with valuable properties for biomedical applications like artificial arteries and other medical implants. However, an environmentally friendly and high productivity process together with the tuning of the mechanical and biological properties of mPHAs are mandatory for this purpose. Here, for the first time, a melt processing technique was applied for the preparation of bionanocomposites starting from poly(3-hydroxyoctanoate) (PHO) and bacterial cellulose nanofibers (BC). The incorporation of only 3 wt % BC in PHO improved its thermal stability with 25 °C and reinforced it, increasing the Young's modulus with 76% and the tensile strength with 44%. The percolation threshold calculated with the aspect ratio of the fibers after melt processing was very low and close to 3 wt %. We showed that this bionanocomposite is able to preserve the ductile behavior during storage, no important aging being noted between 3 h and one month after compression-molding. Moreover, this study is the first to investigate the melt processability of PHO nanocomposite for tube extrusion. In addition, biocompatibility study showed no proinflammatory immune response and better cell adhesion for PHO/BC nanocomposite with 3 wt % BC and demonstrated the high feasibility of this bionanocomposite for in vivo application of tissue-engineered blood vessels.

Biocompatible polyhydroxyalkanoates/bacterial cellulose composites: Preparation, characterization, and in vitro evaluation.

Biocompatible composites play a critical role as scaffolds in tissue engineering. Novel biocomposites made from poly(3-hydroxybutyrate) (PHB), polyhydroxyalkanoate (PHA) and bacterial cellulose (BC) in different concentrations were prepared by solution casting and their thermal and mechanical behavior as well as biocompatibility was characterized. BC addition increased the thermal stability of the polymer matrix as evidenced by thermogravimetric analysis. The crystallinity of PHB and the crystallization temperature decreased with the addition of BC and PHA, thus increasing the processing window. BC in small concentration determined an increase in the mechanical properties due to a concerted action of PHA and filler. Good cells attachment and proliferation were observed for all the biocomposites. By the addition of PHA (more hydrophobic than the matrix) and various amounts of BC (highly hydrophilic), surface properties and cell attachment can be controlled. Cytocompatibility studies using L929 cell line revealed that this material is suitable for biomedical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2576-2584, 2016.

Enhancing antimicrobial activity of TiO2/Ti by torularhodin bioinspired surface modification.

Implant-associated infections are a major cause of morbidity and mortality. This study was performed using titanium samples coated by anodization with a titanium dioxide (TiO2) shielded nanotube layer. TiO2/Ti surface was modified by simple immersion in torularhodin solution and by using a mussel-inspired method based on polydopamine as bio adhesive for torularhodin immobilization. SEM analysis revealed tubular microstructures of torularhodin and the PDA ability to function as a catchy anchor between torularhodin and TiO2 surface. Corrosion resistance was associated with TiO2 barrier oxide layer and nano-organized oxide layer and the torularhodin surface modification does not bring significant changes in resistance of the oxide layer. Our results demonstrated that the torularhodin modified TiO2/Ti surface could effectively prevent adhesion and proliferation of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, and Pseudomonas aeruginosa. The new modified titanium surface showed good biocompatibility and well-behaved haemocompatibility. This biomaterial with enhanced antimicrobial activity holds great potential for future biomedical applications.

Technology transfer of oil-in-water emulsion adjuvant manufacturing for pandemic influenza vaccine production in Romania: Preclinical evaluation of split virion inactivated H5N1 vaccine with adjuvant.

Millions of seasonal and pandemic influenza vaccine doses containing oil-in-water emulsion adjuvant have been administered in order to enhance and broaden immune responses and to facilitate antigen sparing. Despite the enactment of a Global Action Plan for Influenza Vaccines and a multi-fold increase in production capabilities over the past 10 years, worldwide capacity for pandemic influenza vaccine production is still limited. In developing countries, where routine influenza vaccination is not fully established, additional measures are needed to ensure adequate supply of pandemic influenza vaccines without dependence on the shipment of aid from other, potentially impacted first-world countries. Adaptation of influenza vaccine and adjuvant technologies by developing country influenza vaccine manufacturers may enable antigen sparing and corresponding increases in global influenza vaccine coverage capacity. Following on previously described work involving the technology transfer of oil-in-water emulsion adjuvant manufacturing to a Romanian vaccine manufacturing institute, we herein describe the preclinical evaluation of inactivated split virion H5N1 influenza vaccine with emulsion adjuvant, including immunogenicity, protection from virus challenge, antigen sparing capacity, and safety. In parallel with the evaluation of the bioactivity of the tech-transferred adjuvant, we also describe the impact of concurrent antigen manufacturing optimization activities. Depending on the vaccine antigen source and manufacturing process, inclusion of adjuvant was shown to enhance and broaden functional antibody titers in mouse and rabbit models, promote protection from homologous virus challenge in ferrets, and facilitate antigen sparing. Besides scientific findings, the operational lessons learned are delineated in order to facilitate adaptation of adjuvant technologies by other developing country institutes to enhance global pandemic influenza preparedness.

Improved antibacterial behavior of titanium surface with torularhodin-polypyrrole film.

The problem of microorganisms attaching and proliferating on implants and medical devices surfaces is still attracting interest in developing research on different coatings based on antibacterial agents. The aim of this work is centered on modifying titanium (Ti) based implants surfaces through incorporation of a natural compound with antimicrobial effect, torularhodin (T), by means of a polypyrrole (PPy) film. This study tested the potential antimicrobial activity of the new coating against a range of standard bacterial strains: Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and Pseudomonas aeruginosa. The morphology, physical and electrochemical properties of the synthesized films were assessed by SEM, AFM, UV-Vis, FTIR and cyclic voltammetry. In addition, biocompatibility of this new coating was evaluated using L929 mouse fibroblast cells. The results showed that PPy-torularhodin composite film acts as a corrosion protective coating with antibacterial activity and it has no harmful effect on cell viability.