Optimization of Enterotoxigenic Escherichia coli (ETEC) Outer Membrane Vesicles Production and Isolation Method for Vaccination Purposes.

The study addresses Enterotoxigenic Escherichia coli (ETEC), a significant concern in low-income countries. Despite its prevalence, there is no licensed vaccine against ETEC. Bacterial vesicle-based vaccines are promising due to their safety and diverse virulence factors. However, cost-effective production requires enhancing vesicle yield while considering altered properties due to isolation methods. The proposed method involves heat treatment and ultrafiltration to recover vesicles from bacterial cultures. Two vesicle types, collected from heat-treated (HT-OMV) or untreated (NT-OMV) cultures, were compared. Vesicles were isolated via ultrafiltration alone ("complete") or with ultracentrifugation ("sediment"). Preliminary findings suggest complete HT-OMV vesicles are suitable for an ETEC vaccine. They express important proteins (OmpA, OmpX, OmpW) and virulence factors (adhesin TibA). Sized optimally (50-200 nm) for mucosal vaccination, they activate macrophages, inducing marker expression (CD40, MHCII, CD80, CD86) and Th1/Th2 cytokine release (IL-6, MCP-1, TNF-α, IL12p70, IL-10). This study confirms non-toxicity in RAW 264.7 cells and the in vivo ability of complete HT-OMV to generate significant IgG2a/IgG1 serum antibodies. Results suggest promise for a cost-effective ETEC vaccine, requiring further research on in vivo toxicity, pathogen-specific antibody detection, and protective efficacy.

Unveiling a New Selenocyanate as a Multitarget Candidate with Anticancer, Antileishmanial and Antibacterial Potential.

Currently, cancer, leishmaniasis and bacterial infections represent a serious public health burden worldwide. Six cinnamyl and benzodioxyl derivatives incorporating selenium (Se) as selenocyanate, diselenide, or selenide were designed and synthesized through a nucleophilic substitution and/or a reduction using hydrides. Ferrocene was also incorporated by a Friedel-Crafts acylation. All the compounds were screened in vitro for their antiproliferative, antileishmanial, and antibacterial properties. Their capacity to scavenge free radicals was also assessed as a first approach to test their antioxidant activity. Benzodioxyl derivatives 2a-b showed cytotoxicity against colon (HT-29) and lung (H1299) cancer cell lines, with IC50 values below 12 µM, and were also fairly selective when tested in nonmalignant cells. Selenocyanate compounds 1-2a displayed potent antileishmanial activity in L. major and L. infantum, with IC50 values below 5 µM. They also exhibited antibacterial activity in six bacterial strains, notably in S. epidermidis with MIC and MBC values of 12.5 µg/mL. Ferrocene-containing selenide 2c was also identified as a potent antileishmanial agent with radical scavenging activity. Remarkably, derivative 2a with a selenocyanate moiety was found to act as a multitarget compound with antiproliferative, leishmanicidal, and antibacterial activities. Thus, the current work showed that 2a could be an appealing scaffold to design potential therapeutic drugs for multiple pathologies.

Immune Response after Skin Delivery of a Recombinant Heat-Labile Enterotoxin B Subunit of Enterotoxigenic Escherichia coli in Mice.

Enterotoxigenic Escherichia coli (ETEC) infections have been identified as a major cause of acute diarrhoea in children in developing countries, associated with substantial morbidity and mortality rates. Additionally, ETEC remains the most common cause of acute diarrhea of international travellers to endemic areas. The heat-labile toxin (LT) is a major virulence factor of ETEC, with a significant correlation between the presence of antibodies against LT and protection in infected patients. In the present work, we constructed a recombinant LTB unit (rLTB) and studied the capacity of this toxoid incorporated in microneedles (rLTB-MN) to induce a specific immune response in mice. MN were prepared from aqueous blends of the polymer Gantrez AN® [poly (methyl vinyl ether-co-maleic anhydride)], which is not cytotoxic and has been shown to possess immunoadjuvant properties. The mechanical and dissolution properties of rLTB-MNs were evaluated in an in vitro Parafilm M® model and in mice and pig skin ex vivo models. The needle insertion ranged between 378 µm and 504 µm in Parafilm layers, and MNs fully dissolved within 15 min of application inside porcine skin. Moreover, female and male BALB/c mice were immunized through ear skin with one single dose of 5 µg·rLTB in MNs, eliciting significant fecal anti-LT IgA antibodies, higher in female than in male mice. Moreover, we observed an enhanced production of IL-17A by spleen cells in the immunized female mice, indicating a mucosal non-inflammatory and neutralizing mediated response. Further experiments will now be required to validate the protective capacity of this new rLTB-MN formulation against this deadly non-vaccine-preventable disease.

Oral Immunogenicity of Enterotoxigenic Escherichia coli Outer Membrane Vesicles Encapsulated into Zein Nanoparticles Coated with a Gantrez® AN-Mannosamine Polymer Conjugate.

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez®-mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

Development of a Bacterial Nanoparticle Vaccine Against Escherichia coli.

Currently, different subunit-based vaccine strategies against enterobacteria are being investigated. Among those, bacterial outer membrane vesicles (OMV) are promising candidates because of their immunogenic properties and safety. In order to develop an effective vaccine against this kind of pathogens, it is important to induce both systemic and mucosal immunity. For that reason, the oral route of administration would be an adequate option; although it still represents a challenge due to the particular and harsh conditions of the gut. To overcome these inconveniences, different strategies have been proposed, including the use of polymeric nanoparticles based on the copolymer between methyl vinyl ether and maleic anhydride (Gantrez AN). In the present work, a simple procedure for the preparation of heat-induced OMV (named as HT) obtained from Enterotoxigenic Escherichia coli (ETEC) loaded into these poly(anhydride) nanoparticles is described.

Vaccine Based on Outer Membrane Vesicles Using Hydrogels as Vaccine Delivery System.

A simple procedure for obtaining outer membrane vesicles from Salmonella enterica and the use of hydrogels as vaccine delivery system is described. A heat treatment in saline solution of whole bacteria rendered the release of outer membrane vesicles containing relevant antigenic components. The immunogenicity of these antigens when administered by the intranasal route may be improved after embedment into hydrogels to increase residence half-time and thus activate the mucosal immune system.

Understanding the basis of transcutaneous vaccine delivery.

Under many circumstances, prophylactic immunizations are considered as the only possible strategy to control infectious diseases. Considerable efforts are typically invested in immunogen selection but, erroneously, the route of administration is not usually a major concern despite the fact that it can strongly influence efficacy. The skin is now considered a key component of the lymphatic system with tremendous potential as a target for vaccination. The purpose of this review is to present the immunological basis of the skin-associated lymphoid tissue, so as to provide understanding of the skin vaccination strategies. Several strategies are currently being developed for the transcutaneous delivery of antigens. The classical, mechanical or chemical disruptions versus the newest approaches based on microneedles for antigen delivery through the skin are discussed herein.

Prevalence of Integrons and Insertion Sequences in ESBL-Producing E. coli Isolated from Different Sources in Navarra, Spain.

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26⁻ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1⁻IS26⁻IS903⁻ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and ß-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.

Maternal Vaccination. Immunization of Sows during Pregnancy against ETEC Infections.

The immunology of pregnancy is an evolving consequence of multiple reciprocal interactions between the maternal and the fetal-placental systems. The immune response must warrant the pregnancy outcome (including tolerance to paternal antigens), but at the same time, efficiently respond to pathogenic challenges. Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in neonatal and recently weaned pigs. This review aims to give an overview of the current rationale on the maternal vaccination strategies for the protection of the newborn pig against ETEC. Newborn piglets are immunodeficient and naturally dependent on the maternal immunity transferred by colostrum for protection-a maternal immunity that can be obtained by vaccinating the sow during pregnancy. Our current knowledge of the interactions between the pathogen strategies, virulence factors, and the host immune system is aiding the better design of vaccination strategies in this particular and challenging host status. Challenges include the need for better induction of immunity at the mucosal level with the appropriate use of adjuvants, able to induce the most appropriate and long-lasting protective immune response. These include nanoparticle-based adjuvants for oral immunization. Experiences can be extrapolated to other species, including humans.