Nanoparticle-Based Secretory Granules Induce a Specific and Long-Lasting Immune Response through Prolonged Antigen Release.

Developing prolonged antigen delivery systems that mimic long-term exposure to pathogens appears as a promising but still poorly explored approach to reach durable immunities. In this study, we have used a simple technology by which His-tagged proteins can be assembled, assisted by divalent cations, as supramolecular complexes with progressive complexity, namely protein-only nanoparticles and microparticles. Microparticles produced out of nanoparticles are biomimetics of secretory granules from the mammalian hormonal system. Upon subcutaneous administration, they slowly disintegrate, acting as an endocrine-like secretory system and rendering the building block nanoparticles progressively bioavailable. The performance of such materials, previously validated for drug delivery in oncology, has been tested here regarding the potential for time-prolonged antigen release. This has been completed by taking, as a building block, a nanostructured version of p30, a main structural immunogen from the African swine fever virus (ASFV). By challenging the system in both mice and pigs, we have observed unusually potent pro-inflammatory activity in porcine macrophages, and long-lasting humoral and cellular responses in vivo, which might overcome the need for an adjuvant. The robustness of both innate and adaptive responses tag, for the first time, these dynamic depot materials as a novel and valuable instrument with transversal applicability in immune stimulation and vaccinology.

Efficient Delivery of Antimicrobial Peptides in an Innovative, Slow-Release Pharmacological Formulation.

Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections.

Lymphocyte infiltration and antitumoral effect promoted by cytotoxic inflammatory proteins formulated as self-assembling, protein-only nanoparticles.

Two human proteins involved in the inflammatory cell death, namely Gasdermin D (GSDMD) and the Mixed Lineage Kinase Domain-Like (MLKL) protein have been engineered to accommodate an efficient ligand of the tumoral cell marker CXCR4, and a set of additional peptide agents that allow their spontaneous self-assembling. Upon production in bacterial cells and further purification, both proteins organized as stable nanoparticles of 46 and 54 nm respectively, that show, in this form, a moderate but dose-dependent cytotoxicity in cell culture. In vivo, and when administered in mouse models of colorectal cancer through repeated doses, the nanoscale forms of tumor-targeted GSDMD and, at a lesser extent, of MLKL promoted CD8+ and CD20+ lymphocyte infiltration in the tumor and an important reduction of tumor size, in absence of systemic toxicity. The potential of these novel pharmacological agents as anticancer drugs is discussed in the context of synergistic approaches to more effective cancer treatments.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview.

By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.

Recombinant vaccines in 2022: a perspective from the cell factory.

The last big outbreaks of Ebola fever in Africa, the thousands of avian influenza outbreaks across Europe, Asia, North America and Africa, the emergence of monkeypox virus in Europe and specially the COVID-19 pandemics have globally stressed the need for efficient, cost-effective vaccines against infectious diseases. Ideally, they should be based on transversal technologies of wide applicability. In this context, and pushed by the above-mentioned epidemiological needs, new and highly sophisticated DNA-or RNA-based vaccination strategies have been recently developed and applied at large-scale. Being very promising and effective, they still need to be assessed regarding the level of conferred long-term protection. Despite these fast-developing approaches, subunit vaccines, based on recombinant proteins obtained by conventional genetic engineering, still show a wide spectrum of interesting potentialities and an important margin for further development. In the 80's, the first vaccination attempts with recombinant vaccines consisted in single structural proteins from viral pathogens, administered as soluble plain versions. In contrast, more complex formulations of recombinant antigens with particular geometries are progressively generated and explored in an attempt to mimic the multifaceted set of stimuli offered to the immune system by replicating pathogens. The diversity of recombinant antimicrobial vaccines and vaccine prototypes is revised here considering the cell factory types, through relevant examples of prototypes under development as well as already approved products.

Time-Prolonged Release of Tumor-Targeted Protein-MMAE Nanoconjugates from Implantable Hybrid Materials.

The sustained release of small, tumor-targeted cytotoxic drugs is an unmet need in cancer therapies, which usually rely on punctual administration regimens of non-targeted drugs. Here, we have developed a novel concept of protein-drug nanoconjugates, which are packaged as slow-releasing chemically hybrid depots and sustain a prolonged secretion of the therapeutic agent. For this, we covalently attached hydrophobic molecules (including the antitumoral drug Monomethyl Auristatin E) to a protein targeting a tumoral cell surface marker abundant in several human neoplasias, namely the cytokine receptor CXCR4. By this, a controlled aggregation of the complex is achieved, resulting in mechanically stable protein-drug microparticles. These materials, which are mimetics of bacterial inclusion bodies and of mammalian secretory granules, allow the slow leakage of fully functional conjugates at the nanoscale, both in vitro and in vivo. Upon subcutaneous administration in a mouse model of human CXCR4+ lymphoma, the protein-drug depots release nanoconjugates for at least 10 days, which accumulate in the tumor with a potent antitumoral effect. The modification of scaffold cell-targeted proteins by hydrophobic drug conjugation is then shown as a novel transversal platform for the design of slow releasing protein-drug depots, with potential application in a broad spectrum of clinical settings.

Antibacterial Activity of T22, a Specific Peptidic Ligand of the Tumoral Marker CXCR4.

CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms.

Functional Characterization of the Cell Division Gene Cluster of the Wall-less Bacterium Mycoplasma genitalium.

It is well-established that FtsZ drives peptidoglycan synthesis at the division site in walled bacteria. However, the function and conservation of FtsZ in wall-less prokaryotes such as mycoplasmas are less clear. In the genome-reduced bacterium Mycoplasma genitalium, the cell division gene cluster is limited to four genes: mraZ, mraW, MG_223, and ftsZ. In a previous study, we demonstrated that ftsZ was dispensable for growth of M. genitalium under laboratory culture conditions. Herein, we show that the entire cell division gene cluster of M. genitalium is non-essential for growth in vitro. Our analyses indicate that loss of the mraZ gene alone is more detrimental for growth of M. genitalium than deletion of ftsZ or the entire cell division gene cluster. Transcriptional analysis revealed a marked upregulation of ftsZ in the mraZ mutant. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics confirmed the overexpression of FtsZ in MraZ-deprived cells. Of note, we found that ftsZ expression was upregulated in non-adherent cells of M. genitalium, which arise spontaneously at relatively high rates. Single cell analysis using fluorescent markers showed that FtsZ localization varied throughout the cell cycle of M. genitalium in a coordinated manner with the chromosome and the terminal organelle (TMO). In addition, our results indicate a possible role for the RNA methyltransferase MraW in the regulation of FtsZ expression at the post-transcriptional level. Altogether, this study provides an extensive characterization of the cell division gene cluster of M. genitalium and demonstrates the existence of regulatory elements controlling FtsZ expression at the temporal and spatial level in mycoplasmas.

Rational engineering of a human GFP-like protein scaffold for humanized targeted nanomedicines.

Green fluorescent protein (GFP) is a widely used scaffold for protein-based targeted nanomedicines because of its high biocompatibility, biological neutrality and outstanding structural stability. However, being immunogenicity a major concern in the development of drug carriers, the use of exogenous proteins such as GFP in clinics might be inadequate. Here we report a human nidogen-derived protein (HSNBT), rationally designed to mimic the structural and functional properties of GFP as a scaffold for nanomedicine. For that, a GFP-like ß-barrel, containing the G2 domain of the human nidogen, has been rationally engineered to obtain a biologically neutral protein that self-assembles as 10nm-nanoparticles. This scaffold is the basis of a humanized nanoconjugate, where GFP, from the well-characterized protein T22-GFP-H6, has been substituted by the nidogen-derived GFP-like HSNBT protein. The resulting construct T22-HSNBT-H6, is a humanized CXCR4-targeted nanoparticle that selectively delivers conjugated genotoxic Floxuridine into cancer CXCR4+ cells. Indeed, the administration of T22-HSNBT-H6-FdU in a CXCR4-overexpressing colorectal cancer mouse model results in an even more efficient selective antitumoral effect than that shown by its GFP-counterpart, in absence of systemic toxicity. Therefore, the newly developed GFP-like protein scaffold appears as an ideal candidate for the development of humanized protein nanomaterials and successfully supports the tumor-targeted nanoscale drug T22-HSNBT-H6-FdU. STATEMENT OF SIGNIFICANCE: Targeted nanomedicine seeks for humanized and biologically neutral protein carriers as alternative of widely used but immunogenic exogenous protein scaffolds such as green fluorescent protein (GFP). This work reports for the first time the rational engineering of a human homolog of the GFP based in the human nidogen (named HSNBT) that shows full potential to be used in humanized protein-based targeted nanomedicines. This has been demonstrated in T22-HSNBT-H6-FdU, a humanized CXCR4-targeted protein nanoconjugate able to selectively deliver its genotoxic load into cancer cells.

Transcriptional response to metal starvation in the emerging pathogen Mycoplasma genitalium is mediated by Fur-dependent and -independent regulatory pathways.

Transition metals participate in numerous enzymatic reactions and they are essential for survival in all living organisms. For this reason, bacterial pathogens have evolved dedicated machineries to effectively compete with their hosts and scavenge metals at the site of infection. In this study, we investigated the mechanisms controlling metal acquisition in the emerging human pathogen Mycoplasma genitalium. We observed a robust transcriptional response to metal starvation, and many genes coding for predicted lipoproteins and ABC-transporters were significantly up-regulated. Transcriptional analysis of a mutant strain lacking a metalloregulator of the Fur family revealed the activation of a full operon encoding a putative metal transporter system and a gene coding for a Histidine-rich lipoprotein (Hrl). We recognized a conserved sequence with dyad symmetry within the promoter region of the Fur-regulated genes. Mutagenesis of the predicted Fur operator within the hrl promoter abrogated Fur- and metal-dependent expression of a reporter gene. Metal starvation still impelled a strong transcriptional response in the fur mutant, demonstrating the existence of Fur-independent regulatory pathways controlling metal homeostasis. Finally, analysis of metal accumulation in the wild-type strain and the fur mutant by ICP-MS revealed an important role of Fur in nickel acquisition.

Activation of σ20-dependent recombination and horizontal gene transfer in Mycoplasma genitalium.

In the human pathogen Mycoplasma genitalium, homologous recombination is under the control of σ20, an alternative sigma factor that boosts the generation of genetic and antigenic diversity in the population. Under laboratory growth conditions, σ20 activation is rare and the factors governing its intermittent activity are unknown. Two σ20-regulated genes, rrlA and rrlB, showed to be important for recombination of homologous DNA sequences in this bacterium. Herein, we demonstrate that rrlA and rrlB code for two small proteins that participate in a feed-forward loop essential for σ20 function. In addition, we identify novel genes regulated by σ20 and show that several non-coding regions, which function as a reservoir for the generation of antigenic diversity, are also activated by this alternative sigma factor. Finally, we reveal that M. genitalium cells can transfer DNA horizontally by a novel mechanism that requires RecA and is facilitated by σ20 over-expression. This DNA transfer system is arguably fundamental for persistence of M. genitalium within the host since it could facilitate a rapid dissemination of successful antigenic variants within the population. Overall, these findings impose a novel conception of genome evolution, genetic variation and survival of M. genitalium within the host.

Commensal-Specific CD4(+) Cells From Patients With Crohn's Disease Have a T-Helper 17 Inflammatory Profile.

BACKGROUND & AIMS: Crohn's disease (CD) has been associated with an altered immune response to commensal microbiota, mostly based on increased seroreactivity to microbial proteins. Although T cells are believed to contribute to the development of CD, little is known about the antigens involved. We investigated the antigen-specificity of T cells isolated from patients with CD. METHODS: We isolated peripheral blood mononuclear cells from 65 patients with CD and 45 healthy individuals (controls). We investigated T-cell reactivity to commensal microbial antigens using proliferation assays (based on thymidine incorporation and carboxyfluorescein succinimidyl ester dilution). Gene expression patterns were determined using microarray and real-time polymerase chain reaction analyses. Cytokines, chemokines, and antibodies were measured by enzyme-linked immunosorbent assay, flow cytometry, or multiplex cytokine assays. Intestinal crypts were obtained from surgical resection specimens of 7 individuals without inflammatory bowel disease. We examined the effects of commensal-specific CD4(+) T cells on primary intestinal epithelial cells from these samples. RESULTS: The bacterial proteins FlaX, A4-fla2, and YidX increased proliferation of CD4(+) T cells isolated from peripheral blood of patients with CD compared with controls. In blood samples from controls, CD4(+) T cells specific for FlaX, A4-fla2, or YidX had a T-helper (Th)1 phenotype; a larger proportion of CD4(+) T cells specific for these proteins in patients with CD had a Th17 phenotype or produced Th1 and Th17 cytokines. When supernatants collected from commensal-specific CD4(+) T cells from patients with CD were applied to healthy intestinal epithelial cells, the epithelial cells increased the expression of the chemokine (C-X-C motif) ligand 1 (CXCL1), CXCL8 and the CC chemokine ligand 20 (CCL20). CONCLUSIONS: A larger proportion of commensal-specific CD4(+) T cells from patients with CD have a Th17 phenotype or produce Th1 and Th17 cytokines, compared with T cells from controls; this might contribute to intestinal inflammation in patients with CD. These cells might be targeted for treatment of CD. The transcriptional data of commensal-specific CD4(+) T cells from healthy individuals and CD patients have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (accession no: GSE70469).