Search Results (98)

Outer-membrane vesicles (OMVs), naturally secreted by Gram-negative bacteria, have gained recognition as a versatile platform for the development of next-generation vaccines. OMVs are essential contributors to bacterial pathogenesis, horizontal gene transfer, cellular communication, the maintenance of bacterial fitness, and quorum sensing. Their intrinsic immunogenicity, adjuvant properties, and scalability establish OMVs as potent tools for combating infectious diseases and cancer. Recent advancements in genetic engineering and biotechnology have further expanded the utility of OMVs, enabling the incorporation of multiple epitopes and antigens from diverse pathogens. These developments address critical challenges such as antigenic variability and co-infections, offering broader immune coverage and cost-effective solutions. This review explores the unique structural and immunological properties of OMVs, emphasizing their capacity to elicit robust immune responses. It critically examines established and emerging engineering strategies, including the genetic engineering of surface-displayed antigens, surface conjugation, glycoengineering, nanoparticle-based OMV engineering, hybrid OMVs, and in situ OMV production, among others. Furthermore, recent advancements in preclinical research on OMV-based vaccines, including synthetic OMVs, OMV-based nanorobots, and nanodiscs, as well as emerging isolation and purification methods, are discussed. Lastly, future directions are proposed, highlighting the potential integration of synthetic biology techniques to accelerate research on OMV engineering. Full article

Bacterial structures formed from the outer membrane and the periplasm components carry biomolecules to expel cellular material and interact with other cells. These outer membrane vesicles (OMVs) can encapsulate bioactive content, which confers OMVs with high potential as alternative drug delivery vehicles or as a platform for novel vaccine development. Single-gene mutants derived from Escherichia coli JC8031 were engineered to further enhance OMV production based on metabolic network modelling and in silico gene knockout design (ΔpoxB, ΔsgbE, ΔgmhA, and ΔallD). Mutants were experimentally obtained by genome editing using CRISPR-Cas9 and tested for OMVs recovery observing an enhanced OMV production in all of them. Lipidomic analysis through LC-ESI-QTOF-MS was performed for OMVs obtained from each engineered strain and compared to the wild-type E. coli JC8031 strain. The lipid profile of OMVs from the wild-type E. coli JC8031 did not change significantly confirmed by multivariate statistical analysis when compared to the mutant strains. The obtained results suggest that the vesicle production can be further improved while the obtained vesicles are not altered in their composition, allowing further study for stability and integrity for use in therapeutic settings. Full article

Brucellosis, caused by Brucella species, remains a significant zoonotic disease affecting both human and animal health worldwide. Among the outer membrane proteins (Omps) of Brucella, Omp16 has emerged as a key immunogenic target with potential applications in vaccine development and diagnostics. Omp16, a lipidated peptidoglycan-associated lipoprotein, stimulates a strong proinflammatory response and is essential for maintaining the bacterial outer membrane integrity and facilitating host cell invasion. This review examines the immunogenic properties of Omp16, its role in Brucella pathogenesis, and its potential as a candidate for vaccine development. We discuss how Omp16-based vaccines, including recombinant proteins, outer membrane vesicles, and viral vector vaccines, have shown promise in providing protection against Brucella infections in animal models. Additionally, Omp16’s utility in diagnostic applications, particularly in enzyme-linked immunosorbent assays (ELISA), offers a reliable method for detecting brucellosis in both humans and animals. Overall, Omp16 represents a crucial antigen with significant potential for advancing both the diagnosis and prevention of brucellosis, offering insights into the next generation of brucellosis vaccines and diagnostic tools. Full article

Self-amplifying RNA-based (saRNA) technology represents the last frontier in using synthetic RNA in vaccinology. Typically, saRNA consists of positive-strand RNA molecules of viral origin (almost exclusively from alphaviruses) where the sequences of structural proteins are replaced with the open reading frame coding the antigen of interest. For in vivo delivery, they are complexed with lipid nanoparticles (LNPs), just like current COVID-19 vaccines based on synthetic messenger RNA (mRNA). Given their ability to amplify themselves inside the cell, optimal intracellular levels of the immunogenic antigen can be achieved by delivering lower amounts of saRNA molecules compared to mRNA-based vaccines. However, the excessive intracellular accumulation of saRNA may represent a relevant drawback since, as already described in alphavirus-infected cells, the recipient cell may react by incorporating excessive RNA molecules into extracellular vesicles (EVs). These EVs can shed and enter neighboring as well as distant cells, where the EV-associated saRNA can start a new replication cycle. This mechanism could lead to an unwanted and unnecessary spread of saRNA throughout the body, posing relevant safety issues. This perspective article discusses the molecular mechanisms through which saRNAs can be transmitted among different cells/tissues. In addition, a simple way to control the possible excessive saRNA intercellular propagation through the co-expression of an EV-anchored protein inhibiting the saRNA replication is proposed. Based on current knowledge, a safety improvement of saRNA-based vaccines appears to be mandatory for their usage in healthy humans. Full article

In this review, we trace the evolution of liquid chromatography from the pioneering work of Tennikova and Svec to the current monolithic polymethacrylate supports for performing liquid chromatography with biological macromolecules and nanoparticles, which offer rapid, high-throughput separations. By using interconnected channels with a tailored channel diameter, monoliths minimize the diffusion limitations typical of particle-based systems. Radial flow designs and optimized channel architectures enable the direct loading of complex biological fluids, reducing the need for sample preparation and optimizing the purification of large biomolecules and nanoparticles such as proteins, nucleic acids, extracellular vesicles, and viruses. Recent work has integrated monoliths into immunoaffinity and enzyme reactor platforms, streamlining analytical workflows and preparative applications in vaccine production and gene therapy. The ongoing advances in monolithic materials, channel geometry, and continuous processing hold promise for even greater efficiency and scalability in future applications. Full article

Salmonella enterica serovar Typhi (S. Typhi) produces outer membrane vesicles (OMVs) that remain comparatively underexplored as potential biotechnological tools. Here, we investigated how hypervesiculating S. Typhi mutants (ΔtolR and ΔdegS) can be engineered to load and deliver the fluorescent reporter protein mCherry, targeting human epithelial cells and the murine immune system. Deletions in tolR and degS led to distinct OMV phenotypes characterized by higher vesicle production and altered cargo composition, underscoring the impact of disrupted membrane integrity and envelope stress on OMV biogenesis. By fusing mCherry with the S. Typhi OmpA signal peptide (SP_ompA), we achieved robust and functionally intact intravesicular packaging in all strains. Flow cytometry and confocal microscopy revealed that the ΔtolR mutant exhibited particularly high cargo loading in the OMV fraction and pronounced mCherry delivery to epithelial cells, highlighting the potential of hypervesiculation to enhance OMV-based protein transport. However, immunization studies in mice showed that wild-type OMVs, despite carrying less mCherry than their hypervesiculating counterparts, induced the strongest anti-mCherry IgG responses. These findings indicate that, at least under these conditions, antigen loading alone is not sufficient to fully determine immunogenicity. Instead, the intrinsic composition or adjuvant-like properties of OMVs play a pivotal role in driving robust immune activation. Our results establish S. Typhi OMVs, especially when genetically modified with a Sec-dependent targeting signal (SP_ompA), as versatile platforms for heterologous protein delivery. Although hypervesiculation facilitates increased protein encapsulation and delivery to epithelial cells, native OMVs appear to better preserve and/or present antigens for effective immunogenic responses in vivo. These insights set the stage for further optimization of S. Typhi OMVs in vaccine development and protein therapeutics, where balancing cargo loading with immunostimulatory features may be key to achieving maximal efficacy. Full article

Cell-derived, nanoscale extracellular vesicles (EVs) have emerged as promising tools in diagnostic, therapeutic, and vaccine applications. Their unique properties including the capability to encapsulate diverse molecular cargo as well as the versatility in surface functionalization make them ideal candidates for safe and effective vehicles to deliver a range of biomolecules including gene editing cassettes, therapeutic proteins, glycans, and glycoconjugate vaccines. In this review, we discuss recent advances in the development of EVs derived from mammalian and bacterial cells for use in a delivery of carbohydrate-based protein therapeutics and vaccines. We highlight key innovations in EVs’ molecular design, characterization, and deployment for treating diseases including Alzheimer’s disease, infectious diseases, and cancers. We discuss challenges for their clinical translation and provide perspectives for future development of EVs within biopharmaceutical research and the clinical translation landscape. Full article

Nano-drug delivery systems provide targeted solutions for addressing various drug delivery challenges, leveraging nanotechnology to enhance drug solubility and permeability. Liposomes, explored for several decades, face hurdles, especially in oral delivery. Bile-acid stabilized vesicles (bilosomes) are flexible lipid vesicles, composed of phospholipids or other surfactants, along with amphiphilic bile salts, and they show superior stability and pharmacokinetic behavior in comparison to conventional vesicular systems (liposomes and niosomes). Bilosomes enhance skin penetration, fluidize the stratum corneum, and improve drug stability. In oral applications, bilosomes overcome drawbacks, offering improved bioavailability, controlled release, and reduced side effects. Vaccines using bilosomes demonstrate efficacy, and bilosomes for intranasal, inhalation, ocular, and buccal applications enhance drug delivery, offering targeted, efficient, and controlled activities. Formulations vary based on active substances and optimization techniques, showcasing the versatility and potential of bilosomes across diverse drug delivery routes. Therefore, the aim of this comprehensive review was to critically explore the state-of-the-art of bilosomes in drug delivery and potential therapeutic applications. Full article

Modernization of existing methods for the delivery of mRNA is vital in advanced therapeutics. Traditionally, mRNA has faced obstacles of poor stability due to enzymatic degradation. This work examines cutting-edge formulation and emerging techniques for safer delivery of mRNA vaccines. Inspired by the success of lipid nanoparticles (LNP) in delivering mRNA vaccines for COVID-19, a variety of other formulations have been developed to deliver mRNA vaccines for diverse infections. The meritorious features of nanoparticle-based mRNA delivery strategies, including LNP, polymeric, dendrimers, polysaccharide-based, peptide-derived, carbon and metal-based, DNA nanostructures, hybrid, and extracellular vesicles, have been examined. The impact of these delivery platforms on mRNA vaccine delivery efficacy, protection from enzymatic degradation, cellular uptake, controlled release, and immunogenicity has been discussed in detail. Even with significant developments, there are certain limitations to overcome, including toxicity concerns, limited information about immune pathways, the need to maintain a cold chain, and the necessity of optimizing administration methods. Continuous innovation is essential for improving delivery systems for mRNA vaccines. Future research directions have been proposed to address the existing challenges in mRNA delivery and to expand their potential prophylactic and therapeutic application. Full article

Antimicrobial resistance (AMR) is a growing global health crisis that threatens the efficacy of antibiotics and modern medical interventions. The emergence of multidrug-resistant (MDR) pathogens, exacerbated by the misuse of antibiotics in healthcare and agriculture, underscores the urgent need for innovative solutions. (1) Background: AMR arises from complex interactions between human, animal, and environmental health, further aggravated by the overuse and inadequate regulation of antibiotics. Conventional treatments are increasingly ineffective, necessitating alternative strategies. Emerging approaches, including bacteriophage therapy, antimicrobial peptides (AMPs), nanotechnology, microbial extracellular vesicles (EVs), and CRISPR-based antimicrobials, provide novel mechanisms that complement traditional antibiotics in combating resistant pathogens. (2) Methods: This review critically analyzes advanced antibacterial strategies in conjunction with systemic reforms such as antimicrobial stewardship programs, the One Health framework, and advanced surveillance tools. These methods can enhance resistance detection, guide interventions, and promote sustainable practices. Additionally, economic, logistical, and regulatory challenges impeding their implementation are evaluated. (3) Results: Emerging technologies, such as CRISPR and nanotechnology, exhibit promising potential in targeting resistance mechanisms. However, disparities in resource distribution and regulatory barriers hinder widespread adoption. Public–private partnerships and sustainable agriculture practices are critical to overcoming these obstacles. (4) Conclusions: A holistic and integrated approach is essential for mitigating the impact of AMR. By aligning innovative therapeutic strategies with global health policies, fostering interdisciplinary collaboration, and ensuring equitable resource distribution, we can develop a sustainable response to this 21st-century challenge. Full article

Background: An emerging trend of mutual convergence between drug-resistant and highly virulent strains of K. pneumoniae has been identified, highlighting the urgent need for the development of novel vaccines. Methods: To delete the target genes and eliminate the plasmids carrying antibiotic resistance genes, CRISPR-Cas9 technology was employed to perform genome editing on a clinically isolated O2 serotype of K. pneumoniae. Subsequently, this strain was utilized as a host to express genes associated with the synthesis of O1 serotype LPSs to construct the recombinant strain capable of simultaneously expressing LPSs of both O1 and O2 serotypes. This recombinant strain was then used as the production strain for the preparation of vaccines based on GMMAs (Generalized Modules for Membrane Antigens), and its biological characteristics were characterized. Finally, the safety and immunogenicity of the vaccine were evaluated using mice as the model animals. Result: a GMMA vaccine characterized by a high yield and low toxicity was gained. Importantly, the lipopolysaccharides (LPSs) of both O1 and O2 serotypes of K. pneumoniae were successfully expressed on the surface of the outer membrane vesicles. Following immunization with the GMMA vaccine, mice were capable of producing antibodies against the GMMA and demonstrated resistance to the invasion of both serotypes of clinically isolated K. pneumoniae. Conclusions: The GMMA vaccine showed significant promise as a bivalent vaccine against K. pneumoniae. Full article

Outer membrane vesicles (OMVs) are double-layered structures of nanoscale lipids released by gram-negative bacteria. They have the same membrane composition and characteristics as primitive cells, which enables them to penetrate cells and tissues efficiently. These OMVs exhibit excellent membrane stability, immunogenicity, safety, and permeability (which makes it easier for them to penetrate into tumour tissue), making them suitable for developing cancer vaccines and drug delivery systems. Recent studies have focused on engineering OMVs to enhance tumour-targeting capabilities, reduce toxicity, and extend circulation time in vivo. This article reviews the latest progress in OMV engineering for tumour treatment and discusses the challenges associated with the use of OMV-based antitumour therapy in clinical practice. Full article

mRNA-based vaccines against the COVID-19 pandemic have propelled the use of nucleic acids for drug delivery. Conventional lipid-based carriers, such as liposomes and nanolipogels, effectively encapsulate and deliver RNA but are hindered by issues such as premature burst release and immunogenicity. To address these challenges, cell membrane-coated nanoparticles offer a promising alternative. We developed a novel nanoparticle system using chitosan methacrylate-tripolyphosphate (CMATPP), which capitalizes on interactions involving membrane proteins at biointerfaces. Ionic crosslinking between chitosan methacrylate and tripolyphosphate facilitates the formation of nanoparticles amenable to coating with red blood cell (RBC) membranes, extracellular vesicles (EVs), and cell-derived nanovesicles (CDNs). Coating CMATPP nanoparticles with RBC membranes effectively mitigated the initial burst release of encapsulated small interfering RNA (siRNA), sustaining controlled release while preserving membrane proteins. This concept was extended to EVs, where CMATPP nanoparticles and CDNs were incorporated into a microfluidic device and subjected to electroporation to create hybrid CDN-CMATPP nanoparticles. Our findings demonstrate that CMATPP nanoparticles are a robust siRNA delivery system with suppressed burst release and enhanced membrane properties conferred by cell or vesicle membranes. Furthermore, the adaptation of the CDN-CMATPP nanoparticle formation in a microfluidic device suggests its potential for personalized therapies using diverse cell sources and increased throughput via automation. This study underscores the versatility and efficacy of CMATPP nanoparticles in RNA delivery, offering a pathway towards advanced therapeutic strategies that utilize biomimetic principles and microfluidic technologies. Full article

Cryptococcus neoformans is an opportunistic fungal pathogen that is a continuous global health concern, especially for immunocompromised populations. The World Health Organization recognized C. neoformans as one of four critical fungal pathogens, thus emphasizing the need for increased research efforts and clinical resource expansion. Currently, there are no fungal vaccines available for clinical use. Exciting new findings in cryptococcal vaccine development have identified whole cell-based and subunit-based vaccinations to help mitigate health risks and make commercialization attainable. Importantly, recent work has focused on how different cryptococcal cell-wall antigens modified in these vaccine candidates allow us to manipulate their immunogenicity to produce a desired long-term protective anti-fungal immune response. In this review, we discuss the different cryptococcal cell immunogens, namely the polysaccharide capsule, glucans, chitin/chitosan, mannoproteins, and extracellular vesicles, and their role in novel cryptococcal vaccination approaches. Additionally, we examine the immunological mechanisms responsible for protection in these vaccine candidates and the similar host response-stimulation pathways induced through different immunogen exposure. Full article

Milk-derived extracellular vesicles (mEVs) are emerging as promising therapeutic candidates due to their unique properties and versatile functions. These vesicles play a crucial role in immunomodulation by influencing macrophage differentiation and cytokine production, potentially aiding in the treatment of conditions such as bone loss, fibrosis, and cancer. mEVs also have the capacity to modulate gut microbiota composition, which may alleviate the symptoms of inflammatory bowel diseases and promote intestinal barrier integrity. Their potential as drug delivery vehicles is significant, enhancing the stability, solubility, and bioavailability of anticancer agents while supporting wound healing and reducing inflammation. Additionally, bovine mEVs exhibit anti-aging properties and protect skin cells from UV damage. As vaccine platforms, mEVs offer advantages including biocompatibility, antigen protection, and the ability to elicit robust immune responses through targeted delivery to specific immune cells. Despite these promising applications, challenges persist, including their complex roles in cancer, effective antigen loading, regulatory hurdles, and the need for standardized production methods. Achieving high targeting specificity and understanding the long-term effects of mEV-based therapies are essential for clinical translation. Ongoing research aims to optimize mEV production methods, enhance targeting capabilities, and conduct rigorous preclinical and clinical studies. By addressing these challenges, mEVs hold the potential to revolutionize vaccine development and targeted drug delivery, ultimately improving therapeutic outcomes across various medical fields. Full article