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Nanostructured recombinant cytokines: A highly stable alternative to short-lived prophylactics
Abstract
Cytokines have been widely used as adjuvants and therapeutic agents in treatments of human diseases. Despite their recognized potential as drugs, the medical use of cytokines has considerable drawbacks, mainly related to their low stability and short half-life. Such intrinsic limitations imply the administration of high doses, often prompting toxicity, undesirable side effects and greater production costs. Here, we describe a new category of mechanically stable nanostructured cytokines (TNFα and CCL4/MIP-1β) that resist harsh physicochemical conditions in vitro (pH and temperature), while maintaining functionality. These bio-functional materials are produced in recombinant cell factories through cost-effective and fully scalable processes. Notably, we demonstrate their prophylactic potential in vivo showing they protect zebrafish from a lethal infection by Pseudomonas aeruginosa.
... Likely, the composition and structured organization of IB components (protein peptidoglycan, DNA, and RNA) make these protein biomaterials excellent immunogens [13]. Moreover, the authors showed that, when the recombinant protein produced was a cytokine such as TNF-α or CCL4, the nanoparticles were able to interact with relevant immune cells and tissues both when intraperitoneally injected or orally administrated and provided better protection levels compared to similar nanoparticles that included proteins without any specific immune function [21]. These conclusions pushed us to test this concept in swine production as an alternative approach to increase piglet resilience during stressful periods and reduce associated antibiotic use. ...
... Lastly, we also found that GFP nanoparticles induced a greater expression of CLDN4 than the PBS control. In a previous study, Torrealba et al. [21] also found unspecific immunogenicity responses in vitro using nanoparticles with control proteins such as iRFP, but in the in vivo studies, they demonstrated that the effect was better using nanoparticles containing immune-relevant proteins, such as cytokines [21]. It should be noted that all cytokine-based nanoparticles produced herein had a low purity of recombinant protein, indicating that the immunogenicity was probably caused by a combination of several components embedded in the IBs. ...
... Lastly, we also found that GFP nanoparticles induced a greater expression of CLDN4 than the PBS control. In a previous study, Torrealba et al. [21] also found unspecific immunogenicity responses in vitro using nanoparticles with control proteins such as iRFP, but in the in vivo studies, they demonstrated that the effect was better using nanoparticles containing immune-relevant proteins, such as cytokines [21]. It should be noted that all cytokine-based nanoparticles produced herein had a low purity of recombinant protein, indicating that the immunogenicity was probably caused by a combination of several components embedded in the IBs. ...
Antimicrobial resistance is a global threat that is worryingly rising in the livestock sector. Among the proposed strategies, immunostimulant development appears an interesting approach to increase animal resilience at critical production points. The use of nanoparticles based on cytokine aggregates, called inclusion bodies (IBs), has been demonstrated as a new source of immunostimulants in aquaculture. Aiming to go a step further, the objective of this study was to produce cytokine nanoparticles using a food-grade microorganism and to test their applicability to stimulate intestinal mucosa in swine. Four cytokines (IL-1β, IL-6, IL-8, and TNF-α) involved in inflammatory response were produced recombinantly in Lactococcus lactis in the form of protein nanoparticles (IBs). They were able to stimulate inflammatory responses in a porcine enterocyte cell line (IPEC-J2) and alveolar macrophages, maintaining high stability at low pH and high temperature. In addition, an in vivo assay was conducted involving 20 piglets housed individually as a preliminary exploration of the potential effects of IL-1β nanoparticles in piglet intestinal mucosa after a 7 d oral administration. The treated animals tended to have greater levels of TNF-α in the blood, indicating that the tested dose of nanoparticles tended to generate an inflammatory response in the animals. Whether this response is sufficient to increase animal resilience needs further evaluation.
... Zebrafish liver (ZFL) and RTGut cell culture ZFL cells (CRL-2643, ATCC) were cultured at 28°C, 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) 4.5 g l −1 glucose, supplemented with 0.01 mg ml −1 insulin, 50 ng ml −1 EGF, 5% (v/v) antibiotic/antimycotic solution, 10% (v/v) heat inactivated fetal bovine serum (FBS) and 0.5% (v/v) heatinactivated trout serum (TS) as described in Torrealba et al. 21 RTGut cells were obtained from Dr. Carolina Tafalla's laboratory and cultured as described by Kawano et al. 22 Briefly, cells were kept at 20°C in Leibovitz's L-15 Medium GlutaMAX™ (Gibco) #31415029), supplemented with heatinactivated FBS 10% (v/v) and 1% antibiotic/antimycotic. ...
... For positive control of uptake, Atto-488 conjugated TNFα was used, as its uptake has been previously characterized by the research group in this cell line. 21 Both dose-response and time-course experiments were performed in triplicate. Post treatment, cells were washed in PBS and incubated at 28°C with 1 mg ml −1 trypsin (Gibco) for 15 min. ...
... This strong trypsinization step aimed to remove PS-NPs attached to the cell surface. 21 Then, two volumes of complete medium were added, and cells were retrieved by centrifugation at 300 × g for 5 min. Pellets were resuspended in PBS for flow cytometry (FACSCalibur BD), and 10 000 events were counted. ...
The presence of small-sized plastic particles in marine and freshwater environments is a global problem but their long-term impact on ecosystems and human health is still far from being understood. Nanoplastics (<1000 nm) could pose a real and uncontrolled ecological challenge due to their smaller size and sharp ability to penetrate living organisms at any trophic level. Few studies evaluate the impact of nanoplastics in vivo on the immune system of aquatic organisms, while most of them assessed the impact on indirect markers of immune response such as regulation of gene expression, ROS production or DNA genotoxicity, among others. Moreover, the study of the effects of nanoplastics on aquatic vertebrate species in vivo is still scarce. In this context, we seek to shed light on the underlying effects of polystyrene nanoplastics (PS-NPs) on the immune response in a model fish species (Danio rerio, zebrafish) after an acute exposure, with a combination of in vitro and in vivo experiments. Our results show that PS-NPs (65 nm) are efficiently taken up by zebrafish liver cells, accumulating mainly in lysosomes. Furthermore, the expression of immune genes presents a synergy when cells were simultaneously exposed to PS-NPs, at a low dose and early time point (12 h) and challenged with a viral stimulus (poly(I:C)). Moreover, zebrafish larvae also internalize PS-NPs, accumulating them in the gut and pancreas. However, at concentrations of up to 50 mg l⁻¹ in an acute exposure (48 h), PS-NPs do not interfere with the survival of the larvae after a lethal bacterial challenge (Aeromonas hydrophila). This study addresses the relevant environmental question of whether a living organism exposed to PS-NPs can cope with a real immune threat. We show that, although PS-NPs can induce an immune response, the survival of zebrafish larvae challenged with a bacterial infection after an acute exposure to PS-NP is not decimated with respect to unexposed larvae.
... The recombinant TNFα from European sea bass (D. labrax) significantly extended the protection of fish against Vibrio anguillarum when orally immunized together with a commercial oral vaccine of sea bass vibriosis (Galindo-Villegas et al., 2013). In prior studies, our recombinant trout TNFα inclusion bodies (IBs TNFα ) have shown promising features, such as no signs of toxicity both in cells and in vivo, high stability under harsh physicochemical conditions (pH and temperature), as well as a significant survival increase when zebrafish, i.p. injected with IBs TNFα , were challenged with Pseudomonas aeruginosa (Torrealba et al., 2016). However, the ability to evoke a mucosal immune response, to survive in the intestine without degradation, and to cross mucosal barriers in order to gain access to APCs and thus induce local and/or systemic responses, has not been fully investigated. ...
... IBs TNFα were produced as described previously (Torrealba et al., 2016). Briefly, E. coli carrying the TNFα expression plasmid was cultured in LB broth with ampicillin (100 μg/ml) at 37°C. ...
... When OD550 reached 0.5, recombinant protein expression was induced with 1 mM isopropyl-D-thiogalactoside (IPTG, Panreac) for 3 h. To purify IBs, enzymatic and mechanical disruption steps, were carried out as detailed in (Torrealba et al., 2016). Finally, samples were centrifuged at 15,000 ×g for 15 min, and pellets containing purified IBs were stored at −80°C. ...
Waterborne pathogens are a constant threat to fish. Fish mucosa are the point of entry for the majority of microbes and are key in mounting an effective immune response to block invasion, colonization and spread. In the search for tools to enhance immune responses we have evaluated nanostructured TNFα protein nanoparticles (IBsTNFα) as potential mucosal adjuvants. IBsTNFα are non-toxic, stable, protein-based biologically active nanomaterials which are cheap to produce in microbial cell factories. Here we test the in vivo uptake of IBsTNFα in zebrafish larvae by bath immersion and in adult zebrafish by oral gavage. We used flow cytometry, histology and confocal microscopy, to show IBsTNFα can be efficiently taken up by the intestinal mucosa. Fluorescently labelled nanoparticles were ingested by 5 dpf larvae and accumulated in the intestine. In time course studies in adult zebrafish we observed the nanoparticles could penetrate the intestinal epithelia, traverse the lamina propria and enter the muscle layer. Gene expression analysis of intestine and spleen shows that some immune-related genes were significantly modulated. Further, we show that intraperitoneal injected IBsTNFα could protect fish against a lethal infection of Mycobacterium marinum administered by intraperitoneal injection. IBsTNFα therefore can have positive effects on infection outcome and show potential to be developed as a mucosal adjuvant for aquaculture.
... As an alternative to overcome the safety problems associated to live attenuated or DNA vaccines, bacterial inclusion bodies (IBs) nanostructured recombinant proteins have been presented as a new option for vaccination (9). IBs are per se strong stimulants of the fish immune system and have a set of characteristics which make them an attractive alternative: they are mechanically stable, production is scalable and costeffective, they are non-toxic biomaterials and are composed of recombinant proteins. ...
... IBs are per se strong stimulants of the fish immune system and have a set of characteristics which make them an attractive alternative: they are mechanically stable, production is scalable and costeffective, they are non-toxic biomaterials and are composed of recombinant proteins. The latter means they are an adaptable prototype, which could be a good platform for vaccination against a wide range of diseases (9,10). Such nanostructured recombinant proteins have already been shown to protect fish against bacterial infection (9). ...
... The latter means they are an adaptable prototype, which could be a good platform for vaccination against a wide range of diseases (9,10). Such nanostructured recombinant proteins have already been shown to protect fish against bacterial infection (9). ...
Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of different sets of genes in response to stimuli, playing an active role in the homeostasis of the fish immune system. Nowadays, vaccination is one of the main ways to control and prevent viral diseases in aquaculture and the development of novel vaccination approaches is a focal point in fish vaccinology. One of the strategies that has recently emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines have already been shown to immunostimulate and protect fish against bacterial infections. To explore the role of RBCs in the immune response to two nanostructured recombinant proteins, TNFα and a G-VHSV protein fragment, we performed different in vitro and in vivo studies. We show for the first time that rainbow trout RBCs are able to endocytose nanostructured TNFα and G-VHSV protein fragment in vitro, despite not being phagocytic cells, and in response to nanostructured TNFα and G-VHSV fragment, the expression of different immune genes could be modulated.
... The attractiveness of IBs as a fish prophylactic is manifold. Their stability at gastrointestinal pH (13) would allow administrating the antigen orally through the feed, avoiding the necessity for vaccine encapsulation and the cost and stress to fish associated with injection. Production in Escherichia coli is achieved in bulk with a simple enzymatic and mechanical purification procedure which minimizes costs (14). ...
... The IB vehicle, a carrier and viral antigen as one biomaterial, should elicit both an innate and adaptive immune response against the target virus in fish. Finally, IBs' stability under lyophilizing conditions and over a range of temperatures (13) indicates their potential as a practical farm product with a lasting shelf life, avoiding the cold chain. ...
... We have already demonstrated the potential of IBs as an immunostimulant for fish, by nanostructuring recombinant cytokines TNF-α and CCL4 and testing them in a bacterial infection model in zebrafish (13). In addition, uptake of the TNF-α IB by intestinal cells was demonstrated in vivo in rainbow trout via oral intubation (13). ...
... The attractiveness of IBs as a fish prophylactic is manifold. Their stability at gastrointestinal pH (13) would allow administrating the antigen orally through the feed, avoiding the necessity for vaccine encapsulation and the cost and stress to fish associated with injection. Production in Escherichia coli is achieved in bulk with a simple enzymatic and mechanical purification procedure which minimizes costs (14). ...
... The IB vehicle, a carrier and viral antigen as one biomaterial, should elicit both an innate and adaptive immune response against the target virus in fish. Finally, IBs' stability under lyophilizing conditions and over a range of temperatures (13) indicates their potential as a practical farm product with a lasting shelf life, avoiding the cold chain. ...
... We have already demonstrated the potential of IBs as an immunostimulant for fish, by nanostructuring recombinant cytokines TNF-α and CCL4 and testing them in a bacterial infection model in zebrafish (13). In addition, uptake of the TNF-α IB by intestinal cells was demonstrated in vivo in rainbow trout via oral intubation (13). ...
In the search for an eminently practical strategy to develop immunostimulants and vaccines for farmed fish, we have devised recombinant viral antigens presented as “nanopellets” (NPs). These are inclusion bodies of fish viral antigenic proteins produced in Escherichia coli. Soluble recombinant proteins are too labile to endure the in vivo environment and maintain full functionality, and therefore require encapsulation strategies. Yet when they are produced as nanostructures, they can withstand the wide range of gastrointestinal pH found in fish, high temperatures, and lyophilization. Moreover, these nanomaterials are biologically active, non-toxic to fish, cost-effective regarding production and suitable for oral administration. Here, we present three versions of NPs formed by antigenic proteins from relevant viruses affecting farmed fish: the viral nervous necrosis virus coat protein, infectious pancreatic necrosis virus viral protein 2, and a viral haemorrhagic septicemia virus G glycoprotein fragment. We demonstrate that the nanoparticles are taken up in vitro by zebrafish ZFL cells and in vivo by intubating zebrafish as a proof of concept for oral delivery. Encouragingly, analysis of gene expression suggests these NPs evoke an antiviral innate immune response in ZFL cells and in rainbow trout head kidney macrophages. They are therefore a promising platform for immunostimulants and may be candidates for vaccines should protection be demonstrated.
... In vivo, up to 75-80% of macrophages isolated from the zebrafish spleen contained IBs 48 h after IB intraperitoneal injection [51]. [87], and in vivo, as immunostimulants (cytokines) [63]. Oral delivery of IBs in mice, and intraperitoneal or intratumoral injection in zebrafish and in mice, do not result in detectable signs of local or systemic toxicity [ 4 2 1 _ T D $ D I F F ] [63,87]. ...
... [87], and in vivo, as immunostimulants (cytokines) [63]. Oral delivery of IBs in mice, and intraperitoneal or intratumoral injection in zebrafish and in mice, do not result in detectable signs of local or systemic toxicity [ 4 2 1 _ T D $ D I F F ] [63,87]. Tumor necrosis factor (TNF)-a IBs, orally administered to zebrafish, were able to establish close contact with relevant sections of the gut for further IB-derived immune protection from bacterial infections [63]. ...
... Oral delivery of IBs in mice, and intraperitoneal or intratumoral injection in zebrafish and in mice, do not result in detectable signs of local or systemic toxicity [ 4 2 1 _ T D $ D I F F ] [63,87]. Tumor necrosis factor (TNF)-a IBs, orally administered to zebrafish, were able to establish close contact with relevant sections of the gut for further IB-derived immune protection from bacterial infections [63]. Functional IBs, when delivering intracellular proteins with potential healing value, can be regarded as a novel type of nanostructured therapeutic agent or nanopill. ...
Bacterial inclusion bodies (IBs) are functional, non-toxic amyloids occurring in recombinant bacteria showing analogies with secretory granules of the mammalian endocrine system. The scientific interest in these mesoscale protein aggregates has been historically masked by their status as a hurdle in recombinant protein production. However, progressive understanding of how the cell handles the quality of recombinant polypeptides and the main features of their intriguing molecular organization has stimulated the interest in inclusion bodies and spurred their use in diverse technological fields. The engineering and tailoring of IBs as functional protein particles for materials science and biomedicine is a good example of how formerly undesired bacterial byproducts can be rediscovered as promising functional materials for a broad spectrum of applications.
... IBs are protein deposits that are generated in recombinant bacteria under overexpression conditions [5]. Their size ranges from 50 to 500 nm, mainly presenting spherical shapes [23][24][25] and high stability [23,[26][27][28][29]. However, some differences in size and surface functional group density have been described in IBs of the same proteins produced by different bacterial systems [26]. ...
... The activity of these protein nanoparticles produced either in E. coli or L. lactis has led to study their applicability in biotechnology, material sciences and medicine, as biocatalysts [30][31][32], scaffolds in tissue engineering [24], immunomodulators [28,29,33], antimicrobial agents [34], and drug delivery systems in cancer therapy [35][36][37]. Besides, IBs can be used as a source of soluble and active protein [5]. ...
Background
Lactic Acid Bacteria such as Lactococcus lactis, Latilactobacillus sakei (basonym: Lactobacillus sakei) and Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) have gained importance as recombinant cell factories. Although it was believed that proteins produced in these lipopolysaccharides (LPS)-free microorganisms do not aggregate, it has been shown that L. lactis produce inclusion bodies (IBs) during the recombinant production process. These protein aggregates contain biologically active protein, which is slowly released, being a biomaterial with a broad range of applications including the obtainment of soluble protein. However, the aggregation phenomenon has not been characterized so far in L. plantarum. Thus, the current study aims to determine the formation of protein aggregates in L. plantarum and evaluate their possible applications.
Results
To evaluate the formation of IBs in L. plantarum, the catalytic domain of bovine metalloproteinase 9 (MMP-9cat) protein has been used as model protein, being a prone-to-aggregate (PTA) protein. The electron microscopy micrographs showed the presence of electron-dense structures in L. plantarum cytoplasm, which were further purified and analyzed. The ultrastructure of the isolated protein aggregates, which were smooth, round and with an average size of 250–300 nm, proved that L. plantarum also forms IBs under recombinant production processes of PTA proteins. Besides, the protein embedded in these aggregates was fully active and had the potential to be used as a source of soluble protein or as active nanoparticles. The activity determination of the soluble protein solubilized from these IBs using non-denaturing protocols proved that fully active protein could be obtained from these protein aggregates.
Conclusions
These results proved that L. plantarum forms aggregates under recombinant production conditions. These aggregates showed the same properties as IBs formed in other expression systems such as Escherichia coli or L. lactis. Thus, this places this LPS-free microorganism as an interesting alternative to produce proteins of interest for the biopharmaceutical industry, which are obtained from the IBs in an important number of cases.
... The use of catalytically active-IBs has been demonstrated for a wide range of different enzymes such as oxidases, reductases, and synthases, among others , and different studies have reported striking recyclability for catalytic IBs (Koszagova et al., 2018), together with higher stability and activity under harsh conditions, like extreme pHs or the presence of organic solvents, than their soluble counterparts (Diener et al., 2016;Kloss et al., 2018a). Apart from their biotechnological applications, IBs have been successfully applied in biomedicine acting as functional scaffolds in tissue engineering (García-Fruitós et al., 2010;Seras-Franzoso et al., 2013;Tatkiewicz et al., 2013), as targeted-delivery agents Unzueta et al., 2018;Céspedes et al., 2019;Pesarrodona et al., 2019), or as immunostimulants (Schetters et al., 2020;Thwaite et al., 2018;Torrealba et al., 2016a, Torrealba et al., 2016b. ...
... IBs composed of cytokines such as TNFα displayed a prophylactic effect in zebrafish, protecting from infection by the pathogen Pseudomonas aeruginosa. This strategy overcomes the limitations associated with the low stability and short half-life of soluble cytokines (Torrealba et al., 2016a) ( Figure 2). ...
The production of recombinant proteins using microbial cell factories is frequently associated with the formation of inclusion bodies (IBs). These proteinaceous entities can be sometimes a reservoir of stable and active protein, might display good biocompatibility, and are produced efficiently and cost-effectively. Thus, these submicrometric particles are increasingly exploited as functional biomaterials for biotechnological and biomedical purposes. The fusion of aggregation-prone sequences to the target protein is a successful strategy to sequester soluble recombinant polypeptides into IBs. Traditionally, the use of these IB-tags results in the formation of amyloid-like scaffolds where the protein of interest is trapped. This amyloid conformation might compromise the protein’s activity and be potentially cytotoxic. One promising alternative to overcome these limitations exploits the coiled-coil fold, composed of two or more α-helices and widely used by nature to create supramolecular assemblies. In this review, we summarize the state-of-the-art of functional IBs technology, focusing on the coiled-coil-assembly strategy, describing its advantages and applications, delving into future developments and necessary improvements in the field.
... The effects of the IB format on the inflammation of a host were also previously studied by Torrealba et al. (2016a) 16 . They have demonstrated that protein nanoparticles could induce inflammation in Zebrafish and act as an adjuvant 16 , and this effect could be even increased through using nanoparticles composed by proteins with a relevant immune function, such as a cytokine 17 . Similar to results herein, Torrealba et al. (2016b) also described a dose-dependent effect when an unspecific protein like GFP was injected in Zebrafish, with a fast-induced immunostimulating effect 17 . ...
... They have demonstrated that protein nanoparticles could induce inflammation in Zebrafish and act as an adjuvant 16 , and this effect could be even increased through using nanoparticles composed by proteins with a relevant immune function, such as a cytokine 17 . Similar to results herein, Torrealba et al. (2016b) also described a dose-dependent effect when an unspecific protein like GFP was injected in Zebrafish, with a fast-induced immunostimulating effect 17 . This is in agreement with other studies performed previously with LPS 18,19 , chitosan 20 , Panax ginseng extracts 21,22 , among others, in which a rapid immunostimulating effect is observed at dry-off. ...
The cow dry period is a non-milking interval where the mammary gland involutes and regenerates to guarantee an optimal milk production in the subsequent lactation. Important bottlenecks such as the high risk of intramammary infections complicate the process. Antibiotics have been routinely used as a preventive treatment but the concerns about potential antibiotic resistance open a new scenario in which alternative strategies have to be developed. Matrix metalloproteinase-9 (MMP-9) is an enzyme able to degrade the extracellular matrix, triggering the involution and immune function of cow mammary gland. We have studied the infusion into the mammary gland of MMP-9 inclusion bodies as protein-based nanoparticles, demonstrating that 1.2 mg of MMP-9 enhanced the involution and immune function of the cow mammary gland. However, the comparison of the effects triggered by the administration of an active and an inactive form of MMP-9 led to conclude that the response observed in the bovine mammary gland was mainly due to the protein format but not to the biological activity of the MMP-9 embedded in the inclusion body. This study provides relevant information on the future use of protein inclusion bodies in cow mammary gland and the role of MMP-9 at dry-off.
... Another explored application is the injection of targeted-IBs for cancer therapy, proving that these nanoparticles are a stable source of releasing functional proteins [5,18,19]. Besides, it has been shown that IBs can perform an active role as adjuvants for vaccination purposes [20,21], but the real influence of the nanocluster format on inflammatory effects of IBs and their influence on immune-related therapies have never been assessed. Therefore, the exploration of protein-based nanoparticles as a new promising therapeutic format has still some uncovered gaps. ...
... It has been previously reported that IBs display intrinsic immunostimulant properties that allow these to be used as adjuvants [20]. IBs composed of immunostimulant proteins, such as cytokines, have been reported to provide zebrafish with a greater protective performance in vivo [21]. However, the interference of the intrinsic properties of IBs with the immune performance of IBs has never been elucidated before, and this may be critical when assessing the therapeutic potential for immunostimulant protein-based IBs. ...
Bacterial inclusion bodies (IBs) are protein-based nanoparticles of a few hundred nanometers formed during recombinant protein production processes in different bacterial hosts. IBs contain active protein in a mechanically stable nanostructured format that has been broadly characterized, showing promising potential in different fields such as tissue engineering, protein replacement therapies, cancer, and biotechnology. For immunomodulatory purposes, however, the interference of the format immunogenic properties—intrinsic to IBs—with the specific effects of the therapeutic protein is still an uncovered gap. For that, active and inactive forms of the catalytic domain of a matrix metalloproteinase-9 (MMP-9 and mutMMP-9, respectively) have been produced as IBs and compared with the soluble form for dermal inflammatory effects in mmp9 knock-out mice. After protein injections in air-pouches in the mouse model, MMP-9 IBs induce local neutrophil recruitment and increase pro-inflammatory chemokine levels, lasting for at least two days, whereas the effects triggered by the soluble MMP-9 format fade out after 3 h. Interestingly, the IB intrinsic effects (mutMMP-9 IBs) do not last more than 24 h. Therefore, it may be concluded that IBs could be used for the delivery of therapeutic proteins, such as immunomodulating proteins while preserving their stability in the specific tissue and without triggering important unspecific inflammatory responses due to the protein format.
... In vivo, Unzueta and co-authors observed that subcutaneously injected tumor-targeted IBs could release the forming protein, cross into the blood stream and accumulate in the tumor for sustained periods [9]. In addition, when intratumorally injected, IBs made of therapeutic proteins, such as Omomyc and p31, induced tumor cells' death [10], while cytokine-based IBs administered intraperitoneally conferred protection to zebrafish model from a lethal bacterial infection [11]. ...
... Many examples have been published during the last decade that prove that IBs are protein-based biomaterials with promising characteristics to be used for cell replacement therapies, for tissue engineering purposes, or even for cancer treatment [3][4][5][6][7][8][9][10][11]24], among other applications. However, their characteristics and their in vitro behavior have never been correlated with their in vivo performance compared with other standard formats. ...
Inclusion bodies (IBs) are protein nanoclusters obtained during recombinant protein production processes, and several studies have demonstrated their potential as biomaterials for therapeutic protein delivery. Nevertheless, IBs have been, so far, exclusively sifted by their biological activity in vitro to be considered in further protein-based treatments in vivo. Matrix metalloproteinase-9 (MMP-9) protein, which has an important role facilitating the migration of immune cells, was used as model protein. The MMP-9 IBs were compared with their soluble counterpart and with MMP-9 encapsulated in polymeric-based micelles (PM) through ionic and covalent binding. The soluble MMP-9 and the MMP-9-ionic PM showed the highest activity values in vitro. IBs showed the lowest activity values in vitro, but the specific activity evolution in 50% bovine serum at room temperature proved that they were the most stable format. The data obtained with the use of an air-pouch mouse model showed that MMP-9 IBs presented the highest in vivo activity compared to the soluble MMP-9, which was associated only to a low and a transitory peak of activity. These results demonstrated that the in vivo performance is the addition of many parameters that did not always correlate with the in vitro behavior of the protein of interest, becoming especially relevant at evaluating the potential of IBs as a protein-based nanomaterial for therapeutic purposes.
... With the development of protein-based mucosal vaccine, the suitable vaccine delivery system is imperative for the successful protection of protein antigens to overcome their degradation by digestive enzymes present in the GI-tract and induction of sufficient mucosal immune responses. Among various potential delivery systems, IBs isolated from E. coli cells are excellent candidates to be used as protein-based delivery system by the oral route, as they are stable under gastrointestinal pH conditions in vivo and have the suitable nanostructure to be taken up by the gut-associated lymphoid tissue (i.e., Peyer's patches) [18,49]. CVB3 as a leading causative agent of viral myocarditis initiates their infections at the mucosal surface of the gastrointestinal. ...
... This is of special importance science sIgA immune response is of special interest in the development of vaccines against pathogenic microorganisms invading mucosal sites [56]. The usefulness of IBs in oral immunization was also demonstrated in other studies, which also showed the efficient uptake by cells of the intestinal mucosa and induced efficient mucosal response [49,57]. As a result of typical immune response, immunization with VP1 IBs are able to efficiently protect mice from CVB3-induce myocarditis. ...
Background
Oral vaccine is highly desired for infectious disease which is caused by pathogens infection through the mucosal surface. The design of suitable vaccine delivery system is ongoing for the antigen protection from the harsh gastric environment and target to the Peyer’s patches to induce sufficient mucosal immune responses. Among various potential delivery systems, bacterial inclusion bodies have been widely used as delivery systems in the field of nanobiomedicine. However, a large number of heterologous complex proteins could be difficult to propagate in E. coli and fusion partners are often used to enhance target protein expression. As a safety concern the fusion protein need to be removed from the target protein to get tag-free protein, especially for the production of protein antigen in vaccinology. Until now, there is no report on how to remove fusion tag from inclusion body particles in vitro and in vivo. Coxsackievirus B3 (CVB3) is a leading causative agent of viral myocarditis and orally protein vaccine is high desired for CVB3-induced myocarditis. In this context, we explored a tag-free VP1 inclusion body nanoparticles production protocol though a truncated Ssp DnaX mini-intein spontaneous C-cleavage in vivo and also exploited the VP1 inclusion bodies as an oral protein nanoparticle vaccine to protect mice against CVB3-induced myocarditis.
Results
We successfully produced the tag-free VP1 inclusion body nanoparticle antigen of CVB3 and orally administrated to mice. The results showed that the tag-free VP1 inclusion body nanoparticles as an effective antigen delivery system targeting to the Peyer’s patches had the capacity to induce mucosal immunity as well as to efficiently protect mice from CVB3 induce myocarditis without any adjuvant. Then, we proposed the use of VP1 inclusion body nanoparticles as good candidate for oral vaccine to against CVB3-induced myocarditis.
Conclusions
Our tag-free inclusion body nanoparticles production procedure is easy and low cost and may have universal applicability to produce a variety of tag-free inclusion body nanoparticles for oral vaccine.
Electronic supplementary material
The online version of this article (10.1186/s12934-019-1115-z) contains supplementary material, which is available to authorized users.
... Although this is a still moderately understood issue, IB formation causes or it is linked to conformational stresses in the producing bacterial cell (Gasser, et al., 2008) and aggregated species are suspected to show some extent of intrinsic toxicity that might negatively affect bacterial cell growth. However, when eukaryotic cells are exposed to IBs in suspension or these particles are administered to whole organisms through different routes, including injection or oral administration, IBs do not appear to be harmful (Garcia , Diez-Gil, et al., 2010, Liovic, et al., 2012, Seras-Franzoso, et al., 2012, Tatkiewicz, et al., 2013, Seras-Franzoso, et al., 2014, Seras-Franzoso, et al., 2014, Cespedes, et al., 2016, Torrealba, et al., 2016, Torrealba, et al., 2016, Unzueta, et al., 2017, Stamm, et al., 2018, Unzueta, et al., 2018. Therefore, IBs have gained interest as intriguing biocompatible protein materials (Villaverde, 2012, Villaverde, et al., 2012, Loo, et al., 2015 that might be substantially different from protein aggregates linked to prion diseases or to degenerative disorders , Hartl, 2017. ...
... Although this is a still moderately understood issue, IB formation causes or it is linked to conformational stresses in the producing bacterial cell (Gasser, et al., 2008) and aggregated species are suspected to show some extent of intrinsic toxicity that might negatively affect bacterial cell growth. However, when eukaryotic cells are exposed to IBs in suspension or these particles are administered to whole organisms through different routes, including injection or oral administration, IBs do not appear to be harmful (Garcia , Diez-Gil, et al., 2010, Liovic, et al., 2012, Seras-Franzoso, et al., 2012, Tatkiewicz, et al., 2013, Seras-Franzoso, et al., 2014, Seras-Franzoso, et al., 2014, Cespedes, et al., 2016, Torrealba, et al., 2016, Torrealba, et al., 2016, Unzueta, et al., 2017, Stamm, et al., 2018, Unzueta, et al., 2018. Therefore, IBs have gained interest as intriguing biocompatible protein materials (Villaverde, 2012, Villaverde, et al., 2012, Loo, et al., 2015 that might be substantially different from protein aggregates linked to prion diseases or to degenerative disorders , Hartl, 2017. ...
Understanding the structure, functionalities and biology of functional amyloids is an issue of emerging interest. Inclusion bodies, namely protein clusters formed in recombinant bacteria during protein production processes, have emerged as unanticipated, highly tunable models for the scrutiny of the physiology and architecture of functional amyloids. Based on an amyloidal skeleton combined with varying amounts of native or native-like protein forms, bacterial inclusion bodies exhibit an unusual arrangement that confers mechanical stability, biological activity and conditional protein release, being thus exploitable as versatile biomaterials. The applicability of inclusion bodies in biotechnology as enriched sources of protein and reusable catalysts, and in biomedicine as biocompatible topographies, nanopills, or mimetics of endocrine secretory granules has been largely validated. Beyond these uses, the dissection of how recombinant bacteria manage the aggregation of functional protein species into structures of highly variable complexity offers insights about unsuspected connections between protein quality (conformational status compatible with functionality) and cell physiology.
... These aggregates can be easily purified [72] and offer interesting features not available in a soluble form. IBs are an active biomaterial that has already been explored in several applications such as cancer [73], biocatalysis [74], tissue regeneration [75] and immunostimulation [76], as they are highly stable protein nanoparticles with slow-release properties [56,57,77]. Recently, two studies have proven that HDPbased IBs are biologically active against different pathogenic bacteria [20,37]. ...
The antimicrobial resistance crisis calls for the discovery and production of new antimicrobials. Host defense peptides (HDPs) are small proteins with potent antibacterial and immunomodulatory activities that are attractive for translational applications, with several already under clinical trials. Traditionally, antimicrobial peptides have been produced by chemical synthesis, which is expensive and requires the use of toxic reagents, hindering the large-scale development of HDPs. Alternatively, HDPs can be produced recombinantly to overcome these limitations. Their antimicrobial nature, however, can make them toxic to the hosts of recombinant production. In this review we explore the different strategies that are used to fine-tune their activities, bioengineer them, and optimize the recombinant production of HDPs in various cell factories.
... ZFL were cultured at 28°C (optimal temperature for culturing zebrash cells 38 ), 5% CO 2 in Dulbecco's modied Eagle's medium (DMEM) with 4.5 g l −1 glucose, supplemented with 0.01 mg ml −1 insulin, 50 ng ml −1 EGF, 5% (v/v) antibiotic/ antimycotic solution, 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 0.5% (v/v) heat-inactivated trout serum (TS) as described in literature. 39 The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) substrate and DMSO were purchased from Sigma-Aldrich. ...
Mesoporous silica nanoparticles (MSN) characterized by large surface area, pore volume, tunable chemistry, and biocompatibility have been widely studied in nanomedicine as imaging and therapeutic carriers. Most of these studies focused on spherical particles. In contrast, mesoporous silica rods (MSR) that are more challenging to prepare have been less investigated in terms of toxicity, cellular uptake, or biodistribution. Interestingly, previous studies showed that silica rods penetrate fibrous tissues or mucus layers more efficiently than their spherical counterparts. Recently, we reported the synthesis of MSR with distinct aspect ratios and validated their use in multiple imaging modalities by loading the pores with maghemite nanocrystals and functionalizing the silica surface with green and red fluorophores. Herein, based on an initial hypothesis of high liver accumulation of the MSR and a future vision that they could be used for early diagnosis or therapy in fibrotic liver diseases; the cytotoxicity and cellular uptake of MSR were assessed in zebrafish liver (ZFL) cells and the in vivo safety and biodistribution was investigated via fluorescence molecular imaging (FMI) and magnetic resonance imaging (MRI) employing zebrafish larvae and rodents. The selection of these animal models was prompted by the well-established fatty diet protocols inducing fibrotic liver in zebrafish or rodents that serve to investigate highly prevalent liver conditions such as non-alcoholic fatty liver disease (NAFLD). Our study demonstrated that magnetic MSR do not cause cytotoxicity in ZFL cells regardless of the rods' length and surface charge (for concentrations up to 50 mg ml −1 , 6 h) and that MSR are taken up by the ZFL cells in large amounts despite their length of ∼1 mm. In zebrafish larvae, it was observed that they could be safely exposed to high MSR concentrations (up to 1 mg ml −1 for 96 h) and that the rods pass through the liver without causing toxicity. The high accumulation of MSR in rodents' livers at short post-injection times (20% of the administered dose) was confirmed by both FMI and MRI, highlighting the utility of the MSR for liver imaging by both techniques. Our results could open new avenues for the use of rod-shaped silica particles in the diagnosis of pathological liver conditions.
... This strategy has been applied in wound healing [30], for subcutaneous [31] or intranasal delivery of antigens [32,33], or for the local [34] or remote [35] delivery of antitumoral proteins, namely pro-apoptotic factors or tumor-targeted microbial toxins, respectively. In veterinary medicine, they have been proved as excellent immunomodulators and nanostructured vaccines [32,[36][37][38][39][40], since minor amounts of bacterial cell wall lipopolysaccharides from the producing bacterial cells, associated to these protein granules, probably have a positive role on the immunoprotection reached [41]. ...
The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids.
... Zebrafish liver (ZFL) cells were cultured at 28°C in 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose, supplemented with 0.01 mg/mL insulin, 50 ng/mL EGF, 5% (v/v) antibiotic/antimycotic solution, 10% (v/v) heat inactivated fetal bovine serum (FBS), and 0.5% (v/v) heat inactivated trout serum (TS) as described in the literature. 51 The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) substrate and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. ...
Abstract
Multifunctional magnetic nanocomposites based on mesoporous silica have a wide range of potential applications in catalysis, biomedicine or sensing. Such particles combine responsiveness to external magnetic fields with other functionalities endowed by the agents loaded inside the pores or conjugated to the particle surface. Different applications might benefit from specific particle morphologies. In the case of biomedical applications, mesoporous silica nanospheres have been extensively studied while nanorods, with a more challenging preparation, have deserved much less attention despite the positive impact on the therapeutic performance shown by seminal studies. Here, we report on a sol-gel synthesis of mesoporous rod-like silica particles of two distinct lengths (1.4 and 0.9 μm) and aspect ratios (4.7 and 2.2) using Pluronic P123 as a structure-directing template and rendering ~1 g of rods per batch. Iron oxide nanoparticles have been synthesized within the pores yielding maghemite (γ-Fe2O3) nanocrystals of elongated shape (~7x5 nm) with a [110] preferential orientation along the rod axis and a superparamagnetic character. The performance of the rods as T2-weighted MRI contrast agents has also been confirmed. In a subsequent step, the mesoporous silica rods were loaded with a cerium compound and their surface, functionalized with fluorophores (fluorescamine and Cyanine5) emitting at = 525 and 730 nm respectively, thus highlighting the possibility of multiple imaging modalities. Biocompatibility of the rods was evaluated in vitro in a zebrafish (Danio rerio) liver cell line (ZFL), results showing that neither long nor short rods with magnetic particles caused cytotoxicity in ZFL cells for concentrations up to 50 µg/ml. We advocate that such nanocomposites can find applications in medical imaging and therapy where the influence of shape on the performance can be also assessed.
... Several nanotechnology-based-approaches have been developed for vaccination, prophylactic and therapeutic purposes, in the context of infectious diseases, and tested in zebrafish models (Table 3). With prophylactic purposes, Torrealba et al. generated inclusion bodies (IBs) containing TNFα or CCL4 [243]. The resulting nanostructures (IBs) showed good stability under different pH conditions (2.5 and 8). ...
New nanoparticles and biomaterials are increasingly being used in biomedical research for drug delivery, diagnostic applications, or vaccines, and they are also present in numerous commercial products, in the environment and workplaces. Thus, the evaluation of the safety and possible therapeutic application of these nanomaterials has become of foremost importance for the proper progress of nanotechnology. Due to economical and ethical issues, in vitro and in vivo methods are encouraged for the testing of new compounds and/or nanoparticles, however in vivo models are still needed. In this scenario, zebrafish (Danio rerio) has demonstrated potential for toxicological and pharmacological screenings. Zebrafish presents an innate immune system, from early developmental stages, with conserved macrophage phenotypes and functions with respect to humans. This fact, combined with the transparency of zebrafish, the availability of models with fluorescently labelled macrophages, as well as a broad variety of disease models offers great possibilities for the testing of new nanoparticles. Thus, with a particular focus on macrophage–nanoparticle interaction in vivo, here, we review the studies using zebrafish for toxicological and biodistribution testing of nanoparticles, and also the possibilities for their preclinical evaluation in various diseases, including cancer and autoimmune, neuroinflammatory, and infectious diseases.
... mykiss) stimulate the innate immune response, similarly to anterior kidney macrophages of the rainbow trout (O. mykiss) and zebrafish (Danio rerio) liver cells, in response to IB TNFα and IB CCL4 (22) as well as IB VP2−IPNV , IB G−frg16−VHSV , and IB C−VNNV (viral nervous necrosis virus, VNNV) (23). In addition, increased expression of the cd83 and mhc II in the rainbow trout (O. ...
Red blood cells (RBCs)—erythrocytes—of Osteichthyes are primarily known for their involvement in the process of gas exchange and respiration. Currently, physiological properties of RCBs in fish should also include their ability to participate in defense processes as part of the innate and adaptive immune mechanisms. In response to viruses, bacteria, and fungi or recombinant nanoparticles, they can modulate expression of genes responsible for immune reactions, influence activity of leukocytes, and produce cytokines, antimicrobial peptides, and paracrine intercellular signaling molecules. Via the complement system (CR1 receptor) and owing to their phagocytic properties (erythrophagocytosis), RBCs of Osteichthyes can eliminate pathogens. In addition, they are probably involved in the immune response as antigen-presenting cells via major histocompatibility complex class II antigens.
... Several works have studied the IBs appealing features in contexts such as cancer [31], tissue regeneration [32], and immunostimulation [33], demonstrating its great potential as a new biomaterial. However, to the best of our knowledge, this is the first study exploring the antimicrobial effect of a multidomain protein embedded in IBs. ...
Background:
Although most of antimicrobial peptides (AMPs), being relatively short, are produced by chemical synthesis, several AMPs have been produced using recombinant technology. However, AMPs could be cytotoxic to the producer cell, and if small they can be easily degraded. The objective of this study was to produce a multidomain antimicrobial protein based on recombinant protein nanoclusters to increase the yield, stability and effectivity.
Results:
A single antimicrobial polypeptide JAMF1 that combines three functional domains based on human α-defensin-5, human XII-A secreted phospholipase A2 (sPLA2), and a gelsolin-based bacterial-binding domain along with two aggregation-seeding domains based on leucine zippers was successfully produced with no toxic effects for the producer cell and mainly in a nanocluster structure. Both, the nanocluster and solubilized format of the protein showed a clear antimicrobial effect against a broad spectrum of Gram-negative and Gram-positive bacteria, including multi-resistant strains, with an optimal concentration between 1 and 10 µM.
Conclusions:
Our findings demonstrated that multidomain antimicrobial proteins forming nanoclusters can be efficiently produced in recombinant bacteria, being a novel and valuable strategy to create a versatile, highly stable and easily editable multidomain constructs with a broad-spectrum antimicrobial activity in both soluble and nanostructured format.
... Specifically, IFN-α, a representative FDA-approved cytokine has been used in the clinic to treat leukemia since 1986, and subsequently, recombinant interleukin-2 (IL-2) has been developed for cancer immunotherapy since 1992 [59][60][61]. However, these cytokines have short half-lives and limited stability, which can be overcome by using NP-based delivery systems [62,63]. NP-based cytokine delivery can overcome limitations of conventional cytokine-based therapy such as short half-life, autoimmune attack and inflammatory immune reaction [64][65][66][67]. ...
Cancer immunotherapy is an attractive treatment option under clinical settings. However, the major challenges of immunotherapy include limited patient response, limited tumor specificity, immune-related adverse events, and immunosuppressive tumor microenvironment. Therefore, nanoparticle (NP)-based drug delivery has been used to not only increase the efficacy of immunotherapeutic agents, but it also significantly reduces the toxicity. In particular, NP-based drug delivery systems alter the pharmacokinetic (PK) profile of encapsulated or conjugated immunotherapeutic agents to targeted cancer cells or immune cells and facilitate the delivery of multiple therapeutic combinations to targeted cells using single NPs. Recently, advanced NP-based drug delivery systems were effectively utilized in cancer immunotherapy to reduce the toxic side effects and immune-related adverse events. Repurposing these NPs as delivery systems of immunotherapeutic agents may overcome the limitations of current cancer immunotherapy. In this review, we focus on recent advances in NP-based immunotherapeutic delivery systems, such as immunogenic cell death (ICD)-inducing drugs, cytokines and adjuvants for promising cancer immunotherapy. Finally, we discuss the challenges facing current NP-based drug delivery systems that need to be addressed for successful clinical application.
... Because of the lack of cysteins in the displayed antigen, the spinycterins described here may be also looked as a method to reduce the generation of low-immunogenicity inclusion bodies during manufacturing very often found when expressing whole heterologous proteins in recombinant bacteria. Although recent results suggested that isolated nanopellets derived from bacterial inclusion bodies may also be immunogenic [54], their practical use would need additional purification steps, losing their bacterial morphology and their adjuvant properties. Most probably, the use of inclusion bodies as fish vaccines will require too high concentrations. ...
This work describes immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so-called spinycterin vehicles consisting of irreversibly DNA-damaged DNA-repair-less Escherichia coli displaying at their surface a downsized VNNV coat antigen. In this work we have i) maximized bacterial expression levels by downsizing the coat protein of VNNV to a fragment (frgC91-220) containing most of its previously determined antigenicity, ii) developed a scalable autoinduction culture media for E.coli based in soybean rather than in casein hydrolysates, iii) enriched surface expression by screening different anchors from several prokaryotic sources (anchor + frgC91-220 recombinant products), iv) preserved frgC91-220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and v) increased safety using a repair-less E.coli strain as chassis for the spinycterins. These spinycterins protected fish against VNNV challenge with partial (Nmistic + frgC91-220) or total (YBEL + frgC91-220) levels of protection, in contrast to fish immunized with frgC91-220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness and required no adjuvants, thus providing potential to further develop VNNV vaccines for sustainable aquaculture.
... Because of the lack of cysteins in the displayed antigen, the spinycterins described here may be also looked as a method to reduce the generation of low-immunogenicity inclusion bodies during manufacturing very often found when expressing whole heterologous proteins in recombinant bacteria. Although recent results suggested that isolated nanopellets derived from bacterial inclusion bodies may also be immunogenic [54], their practical use would need additional purification steps, losing their bacterial morphology and their adjuvant properties. Most probably, the use of inclusion bodies as fish vaccines will require too high concentrations. ...
This work describes practical immunization of European sea bass (Dicentrarchus labrax) juveniles against viral nervous necrosis virus (VNNV), a betanodavirus causing worldwide mortalities in many fish species. Protection was obtained with the so called spinycterin vehicles consisting in irreversibly DNA-damaged DNA-repair-less E.coli displaying at their surface a downsized antigen. In this work we, i) maximized bacterial expression levels by downsizing the C protein to a fragment (frgC91-220) containing most of its antigenicity, ii) developed an scalable autoinduction bacterial media based in soy-bean increasing membrane display and reproducibility, iii) enriched surface expression by screening different anchors from several prokaryotic origins (anchor+frgC91-220), iv) preserved frgC91-220 antigenicity by inactivating bacteria by irreversible DNA-damage by means of Ciprofloxacin, and v) increased safety using a repair-less E.coli strain as spinycterin chassis. These second generation of spinycterins protected fish against VNNV challenge with partial (Nmistic+frgC91-220) or 100 % (YBEL+frgC91-220 ) protection, in contrast to those fish immunized with frgC91-220 spinycterins. The proposed spinycterin platform has high levels of environmental safety and cost effectiveness, thus providing potential for small fish vaccines for sustainable aquaculture.
... IBs contain functional protein that can be released along time under the appropriate conditions [33]. These features have been previously described and exploited rendering promising results in terms of protein stability and biocompatibility in both, in vitro and in vivo approaches, when a given protein is administered packed as IBs [7,39,44]. Nevertheless, nanostructured protein release from IBs has never been described. ...
Sustained release of drug delivery systems (DDS) has the capacity to increase cancer treatment efficiency in terms of drug dosage reduction and subsequent decrease of deleterious side effects. In this regard, many biomaterials are being investigated but none offers morphometric and functional plasticity and versatility comparable to protein-based nanoparticles (pNPs). Here we describe a new DDS by which pNPs fabricated as bacterial inclusion bodies (IB) can be easily isolated, subcutaneously injected and used as reservoirs for the sustained release of targeted pNPs. Our approach combines the high performance of pNP, regarding specific cell targeting and biodistribution with the IB supramolecular organization, stability and cost effectiveness. This renders a platform able to provide a sustained source of CXCR4-targeted pNPs that selectively accumulate in tumor cells in a CXCR4+ colorectal cancer xenograft model. In addition, the proposed system could be potentially adapted to any other protein construct offering a plethora of possible new therapeutic applications in nanomedicine.
... [1] Moreover, proteinbased materials can be tuned by modifying both production conditions and genetic cell background, [5][6][7][8] to generate a wide spectrum of protein nanoparticles with different composition, structure, size, and shape adapted to specific uses in animal and human medicine. [9][10][11][12] During the last years, peptide-and protein-based biomaterials have emerged as convenient nano-and micro-carriers fully compatible with cells, tissues, and entire organisms. [13] They have great loading capacity and a high efficiency in differential transport, biodistribution, and/or release of compounds with therapeutic interest. ...
The preparation of biological samples for electron microscopy is material- and time-consuming because it is often based on long protocols that also may produce artifacts. Protein labeling for transmission electron microscopy (TEM) is such an example, taking several days. However, for protein-based nanotechnology, high resolution imaging techniques are unique and crucial tools for studying the spatial distribution of these molecules, either alone or as components of biomaterials. In this paper, we tested 2 new short methods of immunolocalization for TEM, and compared them with a standard protocol in qualitative and quantitative approaches by using four protein-based nanoparticles. We reported a significant increase of labeling per area of nanoparticle in both new methodologies (H=19.811; p<0.001) with all the model antigens tested: GFP (H=22.115; p<0.001), MMP-2 (H=19.579; p<0.001), MMP-9 (H=7.567; p<0.023), and IFN-γ(H=62.110; p<0.001). We also found that the most suitable protocol for labeling depends on the nanoparticle's tendency to aggregate. Moreover, the shorter methods reduce artifacts, time (by 30 %), residues and reagents hindering, losing, or altering antigens, and obtaining a significant increase of protein localization (of about 200 %). Overall, this study makes a step forward in the development of optimized protocols for the nanoscale localization of peptides and proteins within new biomaterials.
... Pioneering research describing active rPOI in IBs was published almost 30 years ago [10,11]. Thus, IBs can be used directly as a product, if the rPOI remains in its bioactive form [12]. IBs can be used as catalysts for enzymatic conversions [13][14][15][16][17][18][19], as a reservoir for the controlled release of pharmaceutical proteins in vivo [20][21][22][23], and as a bait for the in vivo immobilization of enzymes for the production of biocatalysts [24,25]. ...
The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.
... Although, some recent results suggest that bacterial inclusion bodies by themselves may also be used as carriers for immunogenic proteins in vaccines, they would also need purification steps. On the other hand, to obtain the necessary immunogenicity level for inclusion bodies may require too high concentrations [43]. ...
Bacterins obtained from recombinant bacteria displaying heterologous antigens in its surface coded by prokaryotic rather than eukaryotic expression plasmids (here called “spiny” bacterins or spinycterins), have been used to increase fish immunogenicity of recombinant viral protein fragments. To explore their immunogenicity, five bacterial-specific membrane anchor-motifs characterized in the literature (Nmistic, Mistic, NTD, YAIN and YBEL) were genetically fused to the immunorelevant cystein-free 117 amino acid fragment II from the ORF149 of cyprinid herpes virus 3 (frgIICyHV3). The fusion of anchor-motifs to the N-terminus of frgIICyHV3 enriched expression in E.coli outer membranes as demonstrated by ELISA, immunofluorescence and flow cytometry of formaldehyde-fixed recombinant bacteria (spinycterins). Unconventional low-intensity ultrasound inducing mucosal micropores in a reversible non-harmful manner was used before carp or zebrafish immersion on spinycterin suspensions as a practical delivery alternative to fish-to-fish injection. After ELISA screening for anti-frgIICyHV3-specific antibodies of spinycterin-immunized fish plasma, the YBEL constructs were identified as the most immunogenic in both carp and zebrafish, correlating with one of the best expressed recombinant proteins as demonstrated by Western blot and surface enriched as demonstrated by ELISA and flow cytometry. The use of prokaryotic expression plasmids to express viral immunorelevant protein fragments in traditionally used fish vaccination bacterins should reduce the environmental concerns raised by DNA vaccination based on eukaryotic expression plasmids. Therefore, spinycterins may be a useful alternative to develop safer fish viral vaccines and mass vaccination methods.
... Nevertheless, IBs have never been explored as protein-based nanoparticles for animal reproduction, enhancers of feed efficiency, or treatment purposes, with only one exception. A recent article described for the first time that IBs formed by cytokines can successfully be used as a prophylactic measure, showing that zebra fish treated with IBs are protected against a lethal infection [192]. In the same way that cytokines have been successfully produced as IBs for prevention purposes in fish, other proteins of interest (hormones, enzymes, and antibodies, among others) or other animal species could also be explored, unfolding enormous possibilities in this field. ...
Recombinant technologies have made possible the production of a broad catalogue of proteins of interest, including those used for animal production. The most widely studied proteins for the animal sector are those with an important role in reproduction, feed efficiency, and health. Nowadays, mammalian cells and fungi are the preferred choice for recombinant production of hormones for reproductive purposes and fibrolytic enzymes to enhance animal performance, respectively. However, the development of low-cost products is a priority, particularly in livestock. The study of cell factories such as yeast and bacteria has notably increased in the last decades to make the new developed reproductive hormones and fibrolytic enzymes a real alternative to the marketed ones. Important efforts have also been invested to developing new recombinant strategies for prevention and therapy, including passive immunization and modulation of the immune system. This offers the possibility to reduce the use of antibiotics by controlling physiological processes and improve the efficacy of preventing infections. Thus, nowadays different recombinant fibrolytic enzymes, hormones, and therapeutic molecules with optimized properties have been successfully produced through cost-effective processes using microbial cell factories. However, despite the important achievements for reducing protein production expenses, alternative strategies to further reduce these costs are still required. In this context, it is necessary to make a giant leap towards the use of novel strategies, such as nanotechnology, that combined with recombinant technology would make recombinant molecules affordable for animal industry.
... Release of functional protein upon internalization of functional IBs by mammalian cells seems to exploit the abundance of properly folded or quasi-folded recombinant protein in these particles, embedded in an amyloid network that represents around 20% of the protein material [12]. Probably assisted by cell chaperones, enzymes [13], growth factors [25], structural proteins [26] and cytokines [27], among others, have been released from internalized IBs and delivered in functional form into receiving cells by using IBs as nanopills. The discovery of IB-like particles in Lactococcus lactis [28,29] (a food-grade microorganism) and the recent generation of IBs in endotoxin-free Escherichia coli strains [30,31] and of functional aggresomes in mammalian cells [32] pave the way for a smoother adaptation of these amyloids to diverse biomedical applications, including therapies. ...
Bacterial inclusion bodies are non-toxic, mechanically stable and functional protein amyloids within the nanoscale size range that are able to naturally penetrate into mammalian cells, where they deliver the embedded protein in a functional form. The potential use of inclusion bodies in protein delivery or protein replacement therapies is strongly impaired by the absence of specificity in cell binding and penetration, thus preventing targeting. To address this issue, we have here explored whether the genetic fusion of two tumor-homing peptides, the CXCR4 ligands R9 and T22, to an inclusion body-forming green fluorescent protein (GFP), would keep the interaction potential and the functionality of the fused peptides and then confer CXCR4 specificity in cell binding and further uptake of the materials. The fusion proteins have been well produced in Escherichia coli in their full-length form, keeping the potential for fluorescence emission of the partner GFP. By using specific inhibitors of CXCR4 binding, we have demonstrated that the engineered protein particles are able to penetrate CXCR4(+) cells, in a receptor-mediated way, without toxicity or visible cytopathic effects, proving the availability of the peptide ligands on the surface of inclusion bodies. Since no further modification is required upon their purification, the biological production of genetically targeted inclusion bodies opens a plethora of cost-effective possibilities in the tissue-specific intracellular transfer of functional proteins through the use of structurally and functionally tailored soft materials.
... In this context, the absence of intrinsic toxicity of bacterial IBs has been widely demonstrated by conventional procedures on IBs formed by diverse proteins with potential biomedical interest, in therapeutic/ prophylactic setting ups. This includes cytoskeleton proteins 50 , enzymes 39 , chaperones 39 and growth factors 42,43,51 among others, and very recently, cytokines in a Zebrafish animal model 52 . ...
In the human endocrine system many protein hormones including urotensin, glucagon, obestatin, bombesin and secretin, among others, are supplied from amyloidal secretory granules. These granules form part of the so called functional amyloids, which within the whole aggregome appear to be more abundant than formerly believed. Bacterial inclusion bodies (IBs) are non-toxic, nanostructured functional amyloids whose biological fabrication can be tailored to render materials with defined biophysical properties. Since under physiological conditions they steadily release their building block protein in a soluble and functional form, IBs are considered as mimetics of endocrine secretory granules. We have explored here if the in vivo implantation of functional IBs in a given tissue would represent a stable local source of functional protein. Upon intratumoral injection of bacterial IBs formed by a potent protein ligand of CXCR4 we have observed high stability and prevalence of the material in absence of toxicity, accompanied by apoptosis of CXCR4⁺ cells and tumor ablation. Then, the local immobilization of bacterial amyloids formed by therapeutic proteins in tumors or other tissues might represent a promising strategy for a sustained local delivery of protein drugs by mimicking the functional amyloidal architecture of the mammals' endocrine system.
Venlafaxine (VFX), a commonly prescribed antidepressant often detected in wastewater effluent, and acute temperature elevations from climate change and increased urbanization, are two environmental stressors currently placing freshwater ecosystems at risk. This study focused on understanding if exposure to VFX impacts the agitation temperature (Tag) and critical thermal maximum (CTmax) of zebrafish (Danio rerio). Additionally, we examined the interactive effects of VFX and acute thermal stress on zebrafish heat shock and inflammatory immune responses. A 96 h 1.0 μg/L VFX exposure experiment was conducted, followed by assessment of thermal tolerance via CTmax challenge. Heat shock proteins and pro-inflammatory immune cytokines were quantified through gene expression analysis by quantitative PCR (qPCR) on hsp 70, hsp 90, hsp 47, il-8, tnfα, and il-1β within gill and liver tissue. No significant changes in agitation temperature between control and exposed fish were observed, nor were there any differences in CTmax based on treatment. Unsurprisingly, hsp 47, 70, and 90 were all upregulated in groups exposed solely to CTmax, while only hsp 47 within gill tissue showed signs of interactive effects, which was significantly decreased in fish exposed to both VFX and CTmax. No induction of an inflammatory response occurred. This study demonstrated that environmentally relevant concentrations of VFX have no impact on thermal tolerance performance in zebrafish. However, VFX can cause diminished function of protective heat shock mechanisms, which could be detrimental to freshwater fish populations and aquatic ecosystems as temperature spikes become more frequent from climate change and urbanization near watersheds.
A broad number of inclusion bodies (IBs) potential uses, including biocatalysis, biocompatible nanomaterials, and nanopills for biomedicine, have been described so far. Recently, it has also been shown that they can also be used as antimicrobial agents. Here, we describe the protocol used to produce and purify IBs with antimicrobial activity at desirable yields and also an optimized and simple methodology to determine the antimicrobial activity of IBs against bacterial strains.Key wordsInclusion bodyAntimicrobialsAntimicrobial activityRecombinant proteinsProtein nanoparticles
In bioprocesses, which target the production of recombinant proteins as inclusion bodies, the upstream process has a decisive influence on the downstream operations, especially regarding cell disruption, inclusion body purity and composition, and refolding yield. Therefore, optimization of the processes in fed-batch mode is a major issue, and screening for strains and process conditions are performed in highly labor, time and cost intensive shake flasks or multiwell plates. Thus, high-throughput experiments performed similar to the industrial operating conditions offer a possibility to develop efficient and robust upstream processes. We present here an automated platform for Escherichia coli fed-batch cultivations in parallelized minibioreactors. The platform allows execution of experiments under multiple conditions while allowing for real-time monitoring of critical process parameters and a controlled fermentation environment. By this, the main factors that affect yields and quality of inclusion bodies can be investigated, speeding up the development process significantly.Key wordsAutomated bioprocess developmentLiquid handling stationHigh throughputFed-batchScreeningInclusion bodies
Escherichia coli
Spring viremia of carp (SVC) remains as a vaccine orphan disease mostly affecting juvenile specimens. Young fish are especially difficult to vaccinate and oral administration of vaccine combined with food would be the election system to minimise stress and the vaccination costs associated to injection. However, administration of prophylactics with food pellets faces off several drawbacks mainly related with vaccine degradation and weak protection correlates of oral vaccines. Here we present a platform based on recombinant proteins (subunit vaccines) manufactured as highly resistant nanostructured materials, and providing excellent levels of protection against SVC virus in a preliminar i.p injection challenge. The G3 domain of SVCV glycoprotein G was overexpressed in E. coli together with IFNγ and the modular protein was purified from bacterial aggregates (inclusion bodies) as highly organised nanostructured biomaterial (nanopellets, NP). These SVCV-IFNNP were taken up by zebrafish cells leading to the enhanced expression of different antiviral and IFN markers (e.g vig1, mx, lmp2 or infgr1 among others) in zebrafish liver cells (ZFL). To monitor if SVCVNP and SVCV-IFNNP can be taken up by intestinal epithelia and can induce antiviral response we performed experiments with SVCVNP and SVCV-IFNNP in 3 days post fertilization (dpf) zebrafish larvae. Both, SVCVNP and SVCV-IFNNP were taken up and accumulated in the intestine without signs of toxicity. The antiviral response in larvae showed a different induction pattern: SVCV-IFNNP did not induce an antiviral response while SVCVNP showed a good antiviral induction. Interestingly ZF4, an embryonic derived cell line, showed an antiviral response like ZFL cells, although the lmp2 and infgr (markers of the IFNγ response) were not overexpressed. Experiments with adult zebrafish indicated an excellent level of protection against a SVCV model infection where SVCV-IFNNP vaccinated fish reached 20% cumulative mortality while control fish reached over 80% cumulative mortality.
Inclusion bodies (IBs) are protein aggregates formed under recombinant protein production processes in microbial cell factories. Their characterization has shown that they are self-assembling and biologically active protein nanoparticles with promising properties for a wide range of applications, including biocatalysis, tissue engineering, and therapy. Besides, different protocols have also been developed to obtain soluble protein from IBs using non-denaturing conditions.KeywordsInclusion bodiesFunctional nanoparticlesProtein nanoparticlesProtein aggregatesRecombinant protein production
Purification of inclusion bodies (IBs) is gaining importance due to the raising of novel applications for these submicron particulate protein clusters, with potential uses in the biomedical and biotechnological fields among others. Here, we present five optimized methods to purify IBs adapting classical procedures to the material nature, as well as the requirements of the producer cell (Gram-negative bacteria, Gram-positive bacteria, or yeast) and the IB final application.Key wordsInclusion BodyPurificationNanoparticlesBacterial cell freeCell disruption
Escherichia coli
Lactococcus lactis
Pichia pastoris
In the last two decades, numerous innovative advances, strategies and protocols have been developed and optimized to improve the quality and quantity of soluble recombinant protein production in E. coli. One of the major challenges being the coelution of chaperone proteins along with desired recombinant protein of interest. The removal of chaperones is important for protein yield, structural determination, optimal activity, and desired function of the recombinant protein. In this chapter, we outline various strategies for removal of chaperone contaminants from oligomeric proteins, with the ultimate objective of ameliorating the quality and proper folding of recombinant proteins. We have discussed in detail the purification and expression of full-length protein, GNE (UDP-N-acetylglucosamine 2-epimerase/ N-acetylmannosamine kinase), as a case study for chaperone removal.
Nervous necrosis virus (NNV) reassortant strains RGNNV/SJNNV have emerged as a potent threat to the Mediterranean marine aquaculture industry, causing viral encephalopathy and retinopathy (VER) in Senegalese sole (Solea senegalensis). In this study, a cheap and practical vaccine strategy using bacterial inclusion bodies made of the coat protein of a virulent reassortant strain of this betanodavirus was devised. The nanostructured recombinant protein nanoparticles, VNNV-CNP, were administered without adjuvant to two groups of juvenile sole, one by intraperitoneal injection and the other by oral intubation. Specific antibodies were raised in vivo against the NNV coat protein via both routes, with a substantial specific antibody expansion in the injected group 30 days post homologous prime boost. Expression levels of five adaptive immune-related genes, cd8a, cd4, igm, igt and arg2, were also quantified in intestine, spleen and head kidney. Results showed cd4 and igm were upregulated in the head kidney of injected fish, indicating activation of an adaptive systemic response, while intubated fish exhibited a mucosal response in the intestine. Neither route showed significant differential expression of cd8a. The specific antibody response elicited in vivo and the lack of any signs of toxicity over the 6-week study period in young fish (n = 100), evidences the potential of the nanoparticle as a vaccine candidate.
Cancer vaccines, which have been widely investigated in the past few decades, are one of the most attractive strategies for cancer immunotherapy. Through the precise delivery of antigens and adjuvants to lymphoid organs or lymphocytes via nanotechnology, innate and adaptive immunity can be boosted to prevent the growth and relapse of malignant tumors. Indeed, nanomedicine offers great opportunities to improve the efficiency of vaccines. Various functional platforms are used to deliver small molecules, peptides, nucleic acids, and even whole cell antigens to the target area of interest, achieving enhanced antitumor immunity and durable therapeutic benefits. Herein, the recent progress in cancer vaccines based on nanotechnology is summarized. Novel platforms used for delivering tumor antigens, promoting adjuvant functions, and combining other therapeutic strategies are discussed. Moreover, possible striving directions and major challenges of nanomedicine for vaccination are also reviewed.
A versatile evaporation-assisted methodology based on the coffee-drop effect is described to deposit nanoparticles on surfaces, obtaining for the first time patterned gradients of protein nanoparticles (pNPs) by using a simple hand-made device. Fully customizable patterns with specific periodicities consisting of stripes with different widths, heights and with distinct nanoparticle concentration gradients can be produced over large areas (~10 cm2) in a fast (up to 1 mm/min), reproducible, and cost-effective manner using an operational protocol optimized by an evolutionary algorithm. The developed method opens the possibility to decorate surfaces “a-la-carte” with pNPs enabling different categories of high throughput studies on cell motility.
Ex vivo gene therapy using retrievable encapsulated cellular implants is an effective strategy for the local and/or chronic delivery of therapeutic proteins. In particular, it is considered an innovative approach to modulate the activity of the immune system. Two recently proposed therapeutic schemes using genetically engineered encapsulated cells are discussed here: the chronic administration of monoclonal antibodies for passive immunization against neurodegenerative diseases and the local delivery of a cytokine as an adjuvant for anti-cancer vaccines.
This review describes the extant knowledge on the teleostean mucosal adaptive immune mechanisms, which is relevant for the development of oral or mucosal vaccines. In the last decade, a number of studies have shed light on the presence of new key components of mucosal immunity: a distinct immunoglobulin class (IgT or IgZ) and the polymeric Ig receptor (pIgR). In addition, intestinal T cells and their putative functions, antigen uptake mechanisms at mucosal surfaces and new mucosal vaccination strategies have been reported. New information on pIgR of Atlantic cod and common carp and comparison of natural and specific cell-mediated cytotoxicity in the gut of common carp and European seabass, is also included in this review. Based on the known facts about intestinal immunology and mucosal vaccination, suggestions are made for the advancement of fish vaccines.
Herein we report the use of immunostimulant-loaded nanoliposomes (called NLcliposomes) as a strategy to protect fish against bacterial and/or viral infections. This work entailed developing a method for in vivo tracking of the liposomes administered to adult zebrafish that enables evaluation of their in vivo dynamics and characterisation of their tissue distribution. The NLc liposomes, which co-encapsulate poly(I:C) and LPS, accumulate in immune tissues and in immunologically relevant cells such as macrophages, as has been assessed in trout primary cell cultures. They protect zebrafish against otherwise lethal bacterial (Pseudomonas aeruginosa PAO1) and viral (Spring Viraemia of Carp Virus) infections regardless of whether they are administered by injection or by immersion, as demonstrated in a series of in vivo infection experiments with adult zebrafish. Importantly, protection was not achieved in fish that had been treated with empty liposomes or with a mixture of the free immunostimulants. Our findings indicate that stimulation of the innate immune system with co-encapsulated immunostimulants in nano-liposomes is a promising strategy to simultaneously improve the levels of protection against bacterial and viral infections in fish.
Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.
Th17 cells are enriched in the gut mucosa and play a critical role in maintenance of the mucosal barrier and host defense
against extracellular bacteria and fungal infections. During chronic human immunodeficiency virus (HIV) infection, Th17 cells
were more depleted compared to Th1 cells, even when the patients had low or undetectable viremia. To investigate the differential
effects of HIV infection on Th17 and Th1 cells, a culture system was used in which CCR6+ CD4+ T cells were sorted from healthy human peripheral blood and activated in the presence of interleukin 1β (IL-1β) and IL-23
to drive expansion of Th17 cells while maintaining Th1 cells. HIV infection of these cultures had minimal effects on Th1 cells
but caused depletion of Th17 cells. Th17 loss correlated with greater levels of virus-infected cells and cell death. In identifying
cellular factors contributing to higher susceptibility of Th17 cells to HIV, we compared Th17-enriched CCR6+ and Th17-depleted CCR6− CD4 T cell cultures and noted that Th17-enriched CCR6+ cells expressed higher levels of α4β7 and bound HIV envelope in an α4β7-dependent manner. The cells also had greater expression
of CD4 and CXCR4, but not CCR5, than CCR6− cells. Moreover, unlike Th1 cells, Th17 cells produced little CCR5 ligand, and transfection with one of the CCR5 ligands,
MIP-1β (CCL4), increased their resistance against HIV. These results indicate that features unique to Th17 cells, including
higher expression of HIV receptors and lack of autocrine CCR5 ligands, are associated with enhanced permissiveness of these
cells to HIV.
Cell factories have been largely exploited for the controlled production of substances of interest for food, pharma and biotech industries. Although human-controlled microbial production and transformation are much older, the cell factory concept was fully established in the 80's through the intensive public and private investment. Strongly empowered by the then nascent recombinant DNA technologies and supported by the approval of recombinant insulin, the principle of controlled biological production as a convenient source of difficult-to-obtain molecules (especially those of high added value) deeply penetrated the industrial tissue, soon becoming a widespread platform aiming at cost-effective large-scale production.
A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics.
We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments.
K14 IBs were electroporated into SW13 cells grown in culture together with a "reporter" plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5.
Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.
The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.
The evolutionarily conserved immune system of the zebrafish (Danio rerio), in combination with its genetic tractability, position it as an excellent model system in which to elucidate the origin and function of vertebrate immune cells. We recently reported the existence of antigen-presenting mononuclear phagocytes in zebrafish, namely macrophages and dendritic cells (DCs), but have been impaired in further characterizing the biology of these cells by the lack of a specific transgenic reporter line. Using regulatory elements of a class II major histocompatibility gene, we generated a zebrafish reporter line expressing green fluorescent protein (GFP) in all APCs, macrophages, DCs, and B lymphocytes. Examination of mhc2dab:GFP; cd45:DsRed double-transgenic animals demonstrated that kidney mhc2dab:GFP(hi); cd45:DsRed(hi) cells were exclusively mature monocytes/macrophages and DCs, as revealed by morphologic and molecular analyses. Mononuclear phagocytes were found in all hematolymphoid organs, but were most abundant in the intestine and spleen, where they up-regulate the expression of inflammatory cytokines upon bacterial challenge. Finally, mhc2dab:GFP and cd45:DsRed transgenes mark mutually exclusive cell subsets in the lymphoid fraction, enabling the delineation of the major hematopoietic lineages in the adult zebrafish. These findings suggest that mhc2dab:GFP and cd45:DsRed transgenic lines will be instrumental in elucidating the immune response in the zebrafish.
The definition of ischaemic stroke has been recently updated as an acute episode of neurological dysfunction caused by focal brain, spinal cord, or retinal ischaemia in the presence of a cerebral infarction. This "tissular" definition has highlighted the importance of pathophysiological processes underlying cerebral damage. In particular, post- ischaemic inflammation in the brain and in the blood stream could influence crucial steps of the tissue injury/repair cascade. CC and CXC chemokines orchestrate the inflammatory response in atherosclerotic plaque vulnerability and cerebral infarction. These molecules exert their activities through the binding to selective transmembrane receptors. CC and CXC chemokines modulate crucial processes (such as inflammatory cell recruitment and activation, neuronal survival, neoangiogenesis). On the other hand, CXC chemokines could also modulate stem cell homing, thus favouring tissue repair. Given this evidence, both CC and CXC chemokines could represent promising therapeutic targets in primary and secondary prevention of ischaemic stroke. Only preliminary studies have been performed investigating treatments with selective chemokine agonists/antagonists. In this review, we will update evidence on the role and the potential therapeutic strategies targeting CC and CXC chemokines in the pathophysiology of ischaemic stroke.
The immune system in the gastrointestinal tract plays a crucial role in the control of infection, as it constitutes the first line of defense against mucosal pathogens. The attractive features of oral immunization have led to the exploration of a variety of oral delivery systems. However, none of these oral delivery systems have been applied to existing commercial vaccines. To overcome this, a new generation of oral vaccine delivery systems that target antigens to gut-associated lymphoid tissue is required. One promising approach is to exploit the potential of microfold (M) cells by mimicking the entry of pathogens into these cells. Targeting specific receptors on the apical surface of M cells might enhance the entry of antigens, initiating the immune response and consequently leading to protection against mucosal pathogens. In this article, we briefly review the challenges associated with current oral vaccine delivery systems and discuss strategies that might potentially target mouse and human intestinal M cells.
Teleost fish are the most primitive bony vertebrates that contain immunoglobulins. In contrast to mammals and birds, these species are devoid of immunoglobulin A (IgA) or a functional equivalent. This observation suggests that specialization of immunoglobulin isotypes into mucosal and systemic responses took place during tetrapod evolution. Challenging that paradigm, here we show that IgT, an immunoglobulin isotype of unknown function, acts like a mucosal antibody. We detected responses of rainbow trout IgT to an intestinal parasite only in the gut, whereas IgM responses were confined to the serum. IgT coated most intestinal bacteria. As IgT and IgA are phylogenetically distant immunoglobulins, their specialization into mucosal responses probably occurred independently by a process of convergent evolution.
Macrophages are phagocytes that have a central role in the organization of the immune system after an infection. These cells can recognize specific molecular components of micro-organisms (pathogen-associated molecular patterns, PAMPs) via specific receptors (PRRs) and elicit specific cellular responses. In the past, the expression of immune genes in response to different PAMPs has been characterized in different fish species. However, little is known about actual cytokine release. We characterized the secretion of tumour necrosis factor (TNF)-α in primary macrophage cultures of rainbow trout (Oncorhynchus mykiss) in response to several PAMPs by Western blot and compared this to the induction of TNF-α gene expression as well as other pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 and anti-viral molecules such as INF-α and Mx protein (Mx). We show that lipopolysaccharide (LPS) and zymosan are major inducers of TNF-α secretion, which is not initially linked to the induction of TNF-α mRNA expression. The secretion of TNF-α, but intriguingly not the expression, is also stimulated by ultrapure LPS meaning that, in fish, contaminants of commercial LPS preparations are better inducers of the inflammatory response. Moreover, we have characterized the signaling pathways that are activated by different PAMPs and the link between those pathways and the final step of TNF-α secretion: TNF-α shedding by TNF-α converting enzyme (TACE/ ADAM17). For the first time, we show that, in fish macrophages, TNF-α is processed by TACE-like activity and this cleavage is dependent upon the activation of ERK, p38MAPK and JNK signaling pathways by LPS.
There exists a unique group of persons who are able to durably control HIV in the absence of therapy. The mechanisms of control in these persons remain poorly defined. In this study, we examined CD8(+) T-cell responses in blood and rectal mucosa from 17 "elite controllers" (viral load < 75 copies/mL), 11 "viremic controllers" (75-2000 copies/mL), 14 noncontrollers (> 10,000 copies/mL), and 10 antiretroviral-treated persons (< 75 copies/mL). Production of interferon-gamma, interleukin-2, tumor necrosis factor-alpha, macrophage inflammatory protein-1 beta, and CD107a by CD8(+) T cells in response to HIV-1 Gag stimulation was measured using flow cytometry. Our hypothesis was that "polyfunctional" T cells producing multiple antiviral factors would be most abundant in mucosal tissues of HIV controllers. Mucosal CD8(+) T-cell responses were significantly stronger and more complex in controllers than in antiretroviral-suppressed persons (P = .0004). The frequency of 4-function responses in rectal mucosa was higher in controllers than in noncontrollers and patients on therapy (P < .0001). Mucosal responses in controllers were frequently stronger and more complex than blood responses. These findings demonstrate that many controllers mount strong, complex HIV-specific T-cell responses in rectal mucosa. These responses may play an important role in mucosal immune surveillance, as suggested by their relative enrichment among persons who control HIV in the absence of therapy.
For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-alpha (Truncated form of Tumor necrosis factor alpha with an N-terminal histidine tag) and dN19 LT-alpha (Truncated form of Lymphotoxin alpha).
Four structurally different proteins that accumulate in the bacterial cell in the form of IBs were studied, revealing that distribution of each target protein between the soluble fraction (cytoplasm) and insoluble fraction (IBs) depends on the nature of the target protein.Irrespective of the folding pattern of each protein, spectroscopy studies have shown that proteins in IBs exhibit similar structural characteristics to the biologically active pure protein when produced at low temperature. In the case of the three studied proteins, G-CSF, His7DeltaN6TNF-alpha, and GFP, a significant amount of protein could be extracted from IBs with 0.2% N-lauroyl sarcosine (NLS) and the proteins retained biological activity although no renaturation procedure was applied.
This study shows that the presence of biologically active proteins inside IBs is more general than usually believed. A large amount of properly folded protein is trapped inside IBs prepared at lower temperatures. This protein can be released from IBs with mild detergents under non-denaturing conditions. Therefore, the active protein can be obtained from such IBs without any renaturation procedure. This is of great importance for the biopharmaceutical industry. Furthermore, such IBs composed of active proteins could also be used as pure nanoparticles in diagnostics, as biocatalysts in enzymatic processes, or even as biopharmaceuticals.
Information on the ontogeny of the fish immune system is largely restricted to a few species of teleosts (e.g., rainbow trout, catfish, zebrafish, sea bass) and has previously focused on morphological features. However, basic questions including the identification of the first lympho-hematopoietic sites, the origin of T- and B-lymphocytes and the acquisition of full immunological capacities remain to be resolved. We review these three main topics with special emphasis on recent results obtained from the zebrafish, a new experimental model particularly suitable for study of the ontogeny of the immune system because of its rapid development and easy manipulation. This species also provides an easy way of creating mutations that can be detected by various types of screens.
The spleen combines the innate and adaptive immune system in a uniquely organized way. The structure of the spleen enables it to remove older erythrocytes from the circulation and leads to the efficient removal of blood-borne microorganisms and cellular debris. This function, in combination with a highly organized lymphoid compartment, makes the spleen the most important organ for antibacterial and antifungal immune reactivity. A better understanding of the function of this complex organ has been gained from recent studies, as outlined in this Review article.
Agents that activate dendritic cells are essential components for vaccines and can be conceptualized as molecular adjuvants. Other molecular adjuvants affect downstream factors that shape the resulting immune response. This review provides a compendium of recently studied molecular adjuvants, focusing on CD8+ T cell responses, which have important roles in HIV vaccines. Reference is also made to CD8+ T cell antitumor responses, where parallel studies of molecular adjuvants are being pursued. Molecular adjuvants can be considered in the following groups: TNF superfamily molecules such as CD40 ligand; agonists for TLRs; agonists for NAIP, CIITA, HET-E, TP-1-leucine-rich repeat pathway receptors, such as nucleotide-binding and oligomerization domain (NOD)1, NOD2, and cryopyrin; chemokines; ILs; CSFs; IFNs; alarmins; and purinergic P2X7 receptor agonists. Complementing these positively acting agents are strategies to reduce the immunosuppressive effects of CD4+CD25+ regulatory T cells and negatively acting factors such as TGF-beta, IL-10, suppressor of cytokine signaling 1, and programmed cell death-1 using neutralizing antibodies, antisense, and small interfering RNA. Especially effective are combinations of molecular adjuvants, which can elicit a massive expansion of antigen-specific CD8+ T cells and show unprecedented efficacy in vaccine and tumor models. Taken together, these new approaches provide significant incremental progress in the development of vaccines to elicit cell-mediated immunity against HIV and other pathogens.
Selective breeding of animals for increased innate resistance offers an attractive strategy to control disease in agriculture. However, this approach is limited by an incomplete knowledge of the heritability, duration, and mechanism(s) of resistance, as well as the impact of selection on the immune response to unrelated pathogens. Herein, as part of a rainbow trout broodstock improvement program, we evaluated factors involved in resistance against a bacterial disease agent, Flavobacterium psychrophilum. In 2005, 71 full-sibling crosses, weighing an average of 2.4 g, were screened, and resistant and susceptible crosses were identified. Naive cohorts were evaluated at 10 and 800 g in size, and most maintained their original relative resistant or susceptible phenotypes, indicating that these traits were stable as size increased >300-fold. During the course of these studies, we observed that the normalized spleen weights of the resistant fish crosses were greater than those of the susceptible fish crosses. To test for direct association, we determined the spleen-somatic index of 103 fish crosses; created high, medium, and low spleen-index groups; and determined survival following challenge with F. psychrophilum or Yersinia ruckeri. Consistent with our previous observations, trout with larger spleen indices were significantly more resistant to F. psychrophilum challenge; however, this result was pathogen-specific, as there was no correlation of spleen size with survival following Y. ruckeri challenge. To our knowledge, this is the first report of a positive association between spleen size and disease resistance in a teleost fish. Further evaluation of spleen index as an indirect measure of disease resistance is warranted.
Fish are subjected to several insults from the environment, which may endanger animal survival. Mucosal surfaces are the first line of defense against these threats, acting as a physical barrier to protect the animal but also functioning as an active immune tissue. Thus, four mucosal-associated lymphoid tissues (MALTs), which lead the immune responses in gut, skin, gills, and nose, have been described in fish. Humoral and cellular immunity, as well as their regulation and the factors that influence the response in these mucosal lymphoid tissues, are still not well known in most fish species. Mucosal B-lymphocytes and immunoglobulins (Igs) are key players in the immune response that takes place in those MALTs. The existence of IgT as a mucosal specialized Ig gives us the opportunity of measuring specific responses after infection or vaccination, a fact that was not possible until recently in most fish species. The vaccination process is influenced by several factors, being stress one of the main stimuli determining the success of the vaccine. Thus, one of the major goals in a vaccination process is to avoid possible situations of stress, which might interfere with fish immune performance. However, interaction between immune and neuroendocrine systems at mucosal tissues is still unknown. In this review, we will summarize the latest findings about B-lymphocytes and Igs in mucosal immunity and the effect of stress and vaccination on B-cell response at mucosal sites. It is important to point out that a limited number of studies have been published regarding stress in mucosa and very few about the influence of stress over mucosal B-lymphocytes.
Objectives
Recombinant protein subunit vaccines are formulated using protein antigens that have been synthesized in heterologous host cells. Several host cells are available for this purpose, ranging from Escherichia coli to mammalian cell lines. This article highlights the benefits of using yeast as the recombinant host.Key findingsThe yeast species, Saccharomyces cerevisiae and Pichia pastoris, have been used to optimize the functional yields of potential antigens for the development of subunit vaccines against a wide range of diseases caused by bacteria and viruses. Saccharomyces cerevisiae has also been used in the manufacture of 11 approved vaccines against hepatitis B virus and one against human papillomavirus; in both cases, the recombinant protein forms highly immunogenic virus-like particles.SummaryAdvances in our understanding of how a yeast cell responds to the metabolic load of producing recombinant proteins will allow us to identify host strains that have improved yield properties and enable the synthesis of more challenging antigens that cannot be produced in other systems. Yeasts therefore have the potential to become important host organisms for the production of recombinant antigens that can be used in the manufacture of subunit vaccines or in new vaccine development.
The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines.
Bacterial inclusion bodies (IBs) have been recently used to generate biocompatible cell culture interfaces with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on FGF-2 IB production and performance has been evaluated. FGF-2 has been overexpressed in Escherichia coli at 25 °C and 37 °C obtaining IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types.
Introduction:
The use of cytokines as therapeutic agents is important, given their potent biological effects. However, this very potency, coupled with the pleiotropic nature and short half-life of these molecules, has limited their therapeutic use. Strategies to increase the half-life and to decrease toxicity are necessary to allow effective treatment with these molecules.
Areas covered:
A number of strategies are used to overcome the natural limitations of cytokines, including PEGylation, encapsulation in liposomes, fusion to targeting peptides or antibodies and latent cytokines. Latent cytokines are engineered using the latency-associated peptide of transforming growth factor-β to produce therapeutic cytokines/peptides that are released only at the site of disease by cleavage with disease-induced matrix metalloproteinases. The principles underlying the latent cytokine technology are described and are compared to other methods of cytokine delivery. The potential of this technology for developing novel therapeutic strategies for the treatment of diseases with an inflammatory-mediated component is discussed.
Expert opinion:
Methods of therapeutic cytokine delivery are addressed. The latent cytokine technology holds significant advantages over other methods of drug delivery by providing simultaneously increased half-life and localised drug delivery without systemic effects. Cytokines that failed clinical trials should be reassessed using this delivery system.
Recombinant technology has ushered in a new era for the pharmaceutical industry. Protein therapeutics, including plasma-derived products and antibodies obtained from the serum of infected patients, have been successfully adopted and utilized to treat various indications. The development of recombinant technology and the subsequent improvement in expression, purification, and formulation technologies have enabled the generation of highly purified proteins in a scalable and cost-effective manner. The discovery and development of several recombinant proteins, such as growth factors and cytokines, will be described followed by a brief review of monoclonal antibodies and enzyme replacement therapy. Recombinant protein-based vaccine, which is the focus of the current review, is described in detail with particular emphasis on several viral and bacterial infections. Challenges and new approaches in their use as a replacement for the currently available vaccines are discussed.
Appropriate combinations of mechanical and biological stimuli are required to promote proper colonization of substrate materials in regenerative medicine. In this context, 3D scaffolds formed by compatible and biodegradable materials are under continuous development in an attempt to mimic the extracellular environment of mammalian cells. We have here explored how novel 3D porous scaffolds constructed by polylactic acid, polycaprolactone or chitosan can be decorated with bacterial inclusion bodies, submicron protein particles formed by releasable functional proteins. A simple dipping-based decoration method tested here specifically favors the penetration of the functional particles deeper than 300 μm from the materials' surface. The functionalized surfaces support the intracellular delivery of biologically active proteins to up to more than 80 % of the colonizing cells, a process that is slightly influenced by the chemical nature of the scaffold. The combination of 3D soft scaffolds and protein-based sustained release systems (Bioscaffolds) offers promise in the fabrication of bio-inspired hybrid matrices for multifactorial control of cell proliferation in tissue engineering under complex architectonic setting-ups.
Recent years have witnessed a renaissance in the study of fish immune systems. Such studies have greatly expanded the knowledge of the evolution and diversification of vertebrate immune systems. Several findings in those studies have overturned old paradigms about the immune system and led to the discovery of novel aspects of mammalian immunity. Here I focus on how findings pertaining to immunity in teleost (bony) fish have led to major new insights about mammalian B cell function in innate and adaptive immunity. Additionally, I illustrate how the discovery of the most ancient mucosal immunoglobulin described thus far will help resolve unsettled paradigms of mammalian mucosal immunity.
Lymphocytes, plasma cells, granulocytes (three to four types), macrophages and monocyte-like cells were ultrastructurally distinguished in the intestinal mucosa of carp. Neutrophilic granulocytes and lymphoid cells were present in and under the epithelium throughout the gut. In contrast to macrophages which dominated in the epithelium of the second segment, basophilic and eosinophilic granulocytes (and their intermediates) were mainly found in the connective tissue of the first segment. Applying monoclonal antibodies against serum immunoglobulin (Ig) in an immunogold technique, only a minority of lymphoid cells appeared to be Ig-immunoreactive at their external membrane, suggesting the presence of many more T than B cells in the intestinal mucosa. Except for cells which resembled immature plasma cells, plasma cells did not show, or hardly showed, Ig at their surface. In contrast with the head kidney, plasma cells with an Ig-immunoreactive cytoplasm were scarce in the intestinal mucosa. As mucosa plasma cells were regularly found with the electron microscope, they possibly contain another class of Ig. Macrophages and monocyte-like cells were also found to be Ig-immunoreactive, suggesting the presence of immune complexes at their external membrane. The immunological significance of B- and T-like lymphocytes next to immune complex-binding and antigen-presenting macrophages in the second gut segment is discussed.
The chemical and mechanical properties of bacterial inclusion bodies, produced in different Escherichia coli genetic backgrounds, have been characterized at the nanoscale level. In regard to wild type, DnaK(-) and ClpA(-) strains produce inclusion bodies with distinguishable wettability, stiffness and stiffness distribution within the proteinaceous particle. Furthermore it was possible to observe how cultured mammalian cells respond differentially to inclusion body variants when used as particulate materials to engineer the nanoscale topography, proving that the actual range of referred mechanical properties is sensed and discriminated by biological systems. The data provide evidence of the mechanistic activity of the cellular quality control network and the regulation of the stereospecific packaging of partially folded protein species in bacteria. This inclusion body nanoscale profiling offers possibilities for their fine genetic tuning and the resulting macroscopic effects when applied in biological interfaces.
Many protein species produced in recombinant bacteria aggregate as insoluble protein clusters named inclusion bodies (IBs). IBs are discarded from further processing or are eventually used as a pure protein source for in vitro refolding. Although usually considered as waste byproducts of protein production, recent insights into the physiology of recombinant bacteria and the molecular architecture of IBs have revealed that these protein particles are unexpected functional materials. In this Opinion article, we present the relevant mechanical properties of IBs and discuss the ways in which they can be explored as biocompatible nanostructured materials, mainly, but not exclusively, in biocatalysis and tissue engineering.