Unveiling the immunomodulatory effect of the novel probiotic Akkermansia muciniphila and its protective effect in vitro.

Akkermansia muciniphila, a bacterium found in the human microbiota, has gained interest due to its potential health benefits. Previous studies have linked its absence to inflammatory disorders, while also suggesting its role in maintaining a healthy gut barrier. However, there is limited information on its specific effects on the immune system. Therefore, the aim of this research was to analyze the in vitro response triggered by A. muciniphila employing RAW 264.7 macrophages. The study focused on investigating the production of cytokines and nitric oxide, along with evaluating the expression of inflammatory surface cellular markers. Additionally, we assessed its potential to protect against intestinal infections, using Salmonella enterica serovar Enteritidis as a model. Our findings reveal a modulation effect of A. muciniphila with pro-inflammatory features, including the release of pro-inflammatory cytokines and upregulation of CD40 and CD80 surface markers, in contrast with previous reported data. Importantly, A. muciniphila could protect against Salmonella infection by promoting macrophage activation, appearing as a promising probiotic candidate for the control of intestinal infections.

Refining Immunogenicity through Intradermal Delivery of Outer Membrane Vesicles against Shigella flexneri in Mice.

Shigellosis remains a global health concern, especially in low- and middle-income countries. Despite improvements in sanitation, the absence of a licensed vaccine for human use has prompted global health organizations to support the development of a safe and effective multivalent vaccine that is cost-effective and accessible for limited-resource regions. Outer Membrane Vesicles (OMVs) have emerged in recent years as an alternative to live attenuated or whole-inactivated vaccines due to their immunogenicity and self-adjuvating properties. Previous works have demonstrated the safety and protective capacity of OMVs against Shigella flexneri infection in mouse models when administered through mucosal or intradermal routes. However, some immunological properties, such as the cellular response or cross-protection among different Shigella strains, remained unexplored. In this study, we demonstrate that intradermal immunization of OMVs with needle-free devices recruits a high number of immune cells in the dermis, leading to a robust cellular response marked by antigen-specific cytokine release and activation of effector CD4 T cells. Additionally, functional antibodies are generated, neutralizing various Shigella serotypes, suggesting cross-protective capacity. These findings highlight the potential of OMVs as a promising vaccine platform against shigellosis and support intradermal administration as a simple and painless vaccination strategy to address this health challenge.

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.

3D printing of microencapsulated Lactobacillus rhamnosus for oral delivery.

3D Printing is an innovative technology within the pharma and food industries that allows the design and manufacturing of novel delivery systems. Orally safe delivery of probiotics to the gastrointestinal tract faces several challenges regarding bacterial viability, in addition to comply with commercial and regulatory standpoints. Lactobacillus rhamnosus CNCM I-4036 (Lr) was microencapsulated in generally recognised as safe (GRAS) proteins, and then assessed for robocasting 3D printing. Microparticles (MP-Lr) were developed and characterised, prior to being 3D printed with pharmaceutical excipients. MP-Lr showed a size of 12.3 ± 4.1 µm and a non-uniform wrinkled surface determined by Scanning Electron Microscopy (SEM). Bacterial quantification by plate counting accounted for 8.68 ± 0.6 CFU/g of live bacteria encapsulated within. Formulations were able to keep the bacterial dose constant upon contact with gastric and intestinal pH. Printlets consisted in oval-shape formulations (15 mm × 8 mm × 3.2 mm) of ca. 370 mg of total weight, with a uniform surface. After the 3D printing process, bacterial viability remained even as MP-Lr protected bacteria alongside the process (log reduction of 0.52, p > 0.05) in comparison with non-encapsulated probiotic (log reduction of 3.05). Moreover, microparticle size was not altered during the 3D printing process. We confirmed the success of this technology for developing an orally safe formulation, GRAS category, of microencapsulated Lr for gastrointestinal vehiculation.

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.

Protection Conferred by Drinking Water Administration of a Nanoparticle-Based Vaccine against Salmonella Enteritidis in Hens.

Salmonellosis remains a major medical and an unmet socioeconomic challenge. Worldwide, more than three million deaths per year are associated with Salmonella enterica serovar Enteritidis infections. Although commercially available vaccines for use in poultry exist, their efficacy is limited. We previously described a method for isolating a heat extract (HE) fraction of the cell surface of S. Enteritidis that contained major antigenic complexes immunogenic in hens naturally infected with the bacterium. One single dose of S. Enteritidis' HE induced protection against lethal salmonellosis in mice. Furthermore, HE encapsulation in nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (PVM/MA), Gantrez AN, improved and prolonged the protection against the disease in mice. We formulated new preparations of Gantrez AN nanoparticles with HE S. Enteritidis and assessed their stability in drinking water and their efficacy in hens after experimental infection. The oral treatment of six-week-old hens with two doses of HE nanoparticles significantly reduced the Salmonella excretion in hens. Due to the effectiveness of the treatment in reducing bacterial excretion, we conclude that HE nanoencapsulation obtained from S. Enteritidis is a viable novel vaccination approach against salmonellosis in farms.

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.

Protective Passive Immunity in Escherichia coli ETEC-Challenged Neonatal Mice Conferred by Orally Immunized Dams with Nanoparticles Containing Homologous Outer Membrane Vesicles.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in mammals, including neonatal, recently weaned pigs and infant human beings. We have previously shown that outer membrane vesicles (OMV) obtained from ETEC serotypes encapsulated into zein nanoparticles, coated with a Gantrez-mannosamine polymer conjugate (OMV-NP), were immunogenic in mice and sows. In the present study, we show that pups from vaccinated mice were protected against ETEC F4 serotype challenge through maternal passive immunization. OMV from F4 cultures were collected and characterized. Two-week-pregnant BALB/c mice were orally immunized with a single dose of vesicles (0.2 mg) either free (OMV) or encapsulated into nanoparticles (OMV-NP). Evaluation of the antibodies in serum (IgG1, Ig2a or IgA) and feces (IgA) of dams immunized with OMV-NP revealed an enhancement of specific immunogenicity. The antibody response conferred by the nanoparticle adjuvant was also correlated with IL-6 and IL-10 splenic levels. Each mother was allowed to feed her progeny for one week. Suckling pups presented specific IgA in feces demonstrating their passive immunization through colostrum intake. Two weeks after the pups were born, they were infected orally with a single dose of F4 E. coli (1.2 × 108 CFU/pup). Results showed that 70% of the pups from dams immunized with OMV-NP were protected. In contrast, 80% of the pups from dams immunized with free OMV died as a result of the experimental challenge. These findings support the use of zein nanoparticles coated with a Gantrez-mannosamine shield as adjuvant delivery system for the oral immunization during pregnancy to confer immunity to the offspring through maternal immunization.

Intranasal delivery system of bacterial antigen using thermosensitive hydrogels based on a Pluronic-Gantrez conjugate.

Thermosensitive hydrogels have been studied as feasible needle-avoidance alternative to vaccine delivery. In this work, we report the development of a new thermal-sensitive hydrogel for intranasal vaccine delivery. This delivery system was formulated with a combination of the polymer Gantrez® AN119 and the surfactant Pluronic® F127 (PF127), with a high biocompatibility, biodegradability and immunoadjuvant properties. Shigella flexneri outer membrane vesicles were used as the antigen model. A stable and easy-to-produce thermosensitive hydrogel which allowed the incorporation of the OMV-antigenic complex was successfully synthetized. A rapid gel formation was achieved at body temperature, which prolonged the OMV-antigens residence time in the nasal cavity of BALB/c mice when compared to intranasal delivery of free-OMVs. In addition, the bacterial antigens showed a fast release profile from the hydrogel in vitro, with a peak at 30 min of incubation at 37 °C. Hydrogels appeared to be non-cytotoxic in the human epithelial HeLa cell line and nose epithelium as well, as indicated by the absence of histopathological features. Immunohistochemical studies revealed that after intranasal administration the OMVs reached the nasal associated lymphoid tissue. These results support the use of here described thermosensitive hydrogels as a potential platform for intranasal vaccination.

Dissolving Microneedles for Intradermal Vaccination against Shigellosis.

Intradermal (ID) immunization is of increasing interest due to the easy accessibility and excellent immunogenic properties of the skin. Among ID immunization methods, dissolving microneedles (MNs) have appeared as an alternative to traditional hypodermic immunization, offering many advantages, such as being an easily administered method, with no need for health personnel, painless, and avoiding the use of needles and sharp wastage. In this study, an affordable and easy-to-produce MNs method was developed based on aqueous blends of 30% w/w poly (methyl vinyl ether-co-maleic anhydride). As an antigen model, a subunit vaccine candidate based on outer membrane vesicles from Shigella flexneri was used. Both unloaded and antigen-loaded MNs were synthetized and characterized. The MNs were successfully validated in an in vitro Parafilm M® skin model and in a pig skin ex vivo model. Biodistribution studies were performed in BALB/c mice using 99mTcO4- radiolabeled samples. Results indicated that the vesicle vaccine was successfully released from the MNs and targeted gastrointestinal tract after 6 h post-administration. In vivo immunization and protection studies were performed in BALB/c mice. Mice were intradermally immunized through ear skin with one single dose of 200 g antigenic complex, eliciting the production of specific systemic IgG and mucosal IgA. Moreover, MNs were able to protect mice from an experimental infection with 1×106 CFU/mouse of S. flexneri four weeks after immunization. This work demonstrates for the first time the potential of outer membrane vesicle-loaded dissolving MNs for ID vaccination against enteropathogens like Shigella.

Mannosylated Nanoparticles for Oral Immunotherapy in a Murine Model of Peanut Allergy.

Peanut allergy is one of the most prevalent and severe of food allergies with no available cure. The aim of this work was to evaluate the potential of an oral immunotherapy based on the use of a roasted peanut extract encapsulated in nanoparticles with immunoadjuvant properties. For this, a polymer conjugate formed by the covalent binding of mannosamine to the copolymer of methyl vinyl ether and maleic anhydride was first synthetized and characterized. Then, the conjugate was used to prepare nanoparticles with an important capability to diffuse through the mucus layer and reach, in a large extent, the intestinal epithelium, including Peyer's patches. Their immunotherapeutic potential was evaluated in a model of presensitized CD1 mice to peanut. After completing therapy, mice underwent an intraperitoneal challenge with peanut extract. Nanoparticle-treatment was associated with both less serious anaphylaxis symptoms and higher survival rates than control, confirming the protective effect of this formulation against the challenge.

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.

Oral Immunogenicity in Mice and Sows of Enterotoxigenic Escherichia Coli Outer-Membrane Vesicles Incorporated into Zein-Based Nanoparticles.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in neonatal and recently weaned pigs. The immune protection of the piglets derives from maternal colostrum, since this species does not receive maternal antibodies through the placenta. In the present study, outer membrane vesicles (OMVs) obtained from main ETEC strains involved in piglet infection (F4 and F18 serotypes), encapsulated into zein nanoparticles coated with Gantrez®® AN-mannosamine conjugate, were used to orally immunize mice and pregnant sows. Loaded nanoparticles were homogeneous and spherical in a shape, with a size of 220-280 nm. The diffusion of nanoparticles through porcine intestinal mucus barrier was assessed by a Multiple Particle Tracking technique, showing that these particles were able to diffuse efficiently (1.3% diffusion coefficient), validating their oral use. BALB/c mice were either orally immunized with free OMVs or encapsulated into nanoparticles (100 µg OMVs/mouse). Results indicated that a single dose of loaded nanoparticles was able to elicit higher levels of serum specific IgG1, IgG2a and IgA, as well as intestinal IgA, with respect to the free antigens. In addition, nanoparticles induced an increase in levels of IL-2, IL-4 and IFN-γ with respect to the administration of free OMVs. Orally immunized pregnant sows with the same formulation elicited colostrum-, serum- (IgG, IgA or IgM) and fecal- (IgA) specific antibodies and, what is most relevant, offspring suckling piglets presented specific IgG in serum. Further studies are needed to determine the infection protective capacity of this new oral subunit vaccine.

Towards a subunit vaccine from a Shigella flexneri ΔtolR mutant.

Disruption of one or more components of the Tol-Pal system, involved in maintaining the integrity of the outer membrane of Gram-negative bacteria, has been proposed as a method to increase the yield obtained from natural production of outer membrane vesicles (OMV). We present a new OMV-based product, obtained from genetically modified Shigella flexneri 2a with a non-polar deletion in tolR and heat-inactivated (HT-ΔtolR). The S. flexneri ΔtolR strain lead to a higher release of vesicles, more than 8-times when compared to the yield obtained from chemically inactivated wild type strain. S. flexneri mutant strain appeared to be more sensitive to different chemical compounds, including antibiotics, bile salts or human complement and it was also less virulent in both in vitro and in vivo assays. The mutation produced some changes in the LPS O-chain and protein expression. S. flexneri ΔtolR was enriched in long and very long LPS O-chain and expressed a different pattern of surface proteins or lipoproteins. In vitro toxicity and activation properties were determined in Raw 267.4 macrophage cell line. HT-ΔtolR antigenic complex was non-cytotoxic and activation markers, such as MHC-II or CD40, were highly expressed during incubation with this product. Finally, preliminary studies on the antibody response elicited by HT-ΔtolR demonstrated a robust and diverse response in mice. Considering these promising results, HT-ΔtolR antigenic extract appears as a new potential vaccine candidate to face shigellosis.

Poly(anhydride) nanoparticles containing cashew nut proteins can induce a strong Th1 and Treg immune response after oral administration.

Cashew nut allergy is the second most commonly reported tree nut allergy. Traditional allergen immunotherapy presents several clinical drawbacks that can be reduced by using nanoparticles-basedallergen-delivery systems, modulating the immune response towards a protective one. In this context, the goal of this work was to assess the potential of poly(anhydride) nanoparticles (NP) for cashew nut oral immunization. Cashew nut allergens-loaded nanoparticles (CNE-NP) were prepared by solvent displacement method. After nanoparticles characterization, oral immunomodulation ability was evaluated in BALB/c mice. Our results demonstrated that CNE-NP induced a higher Th1/Th2 ratio in comparison with animals immunized with free cashew nut proteins. Indeed, a decrease in splenic Th2 cytokines (IL-4, IL-5, and IL-13), and an enhancement of pro-Th1 (IL-12 and IFN-γ) and regulatory (IL-10) cytokines was observed. Furthermore, mice orally immunized with CNE-NP presented an increased expansion of CD4+ T regulatory cells, such as CD4+Foxp3+ and CD4+LAP+, in the mesenteric lymph nodes. In conclusion, oral immunization with a single dose of poly(anhydride) nanoparticles loaded with cashew nut proteins leaded to a pro-Th1 and Treg immune response. Furthermore, their immunomodulatory properties could be introduced as a new approach for management of cashew nut allergy.

New pharmaceutical approaches for the treatment of food allergies.

INTRODUCTION: Allergic diseases constitute one of the most common causes of chronic illness in developed countries. The main mechanism determining allergy is an imbalance between Th1 and Th2 response towards Th2. AREAS COVERED: This review describes the mechanisms underlying the natural tolerance to food components and the development of an allergic response in sensitized individuals. Furthermore, therapeutic approaches proposed to manage these abnormal immunologic responses food are also presented and discussed. EXPERT OPINION: In the past, management of food allergies has consisted of the education of patients to avoid the ingestion of the culprit food and to initiate the therapy (e.g. self-injectable epinephrine) in case of accidental ingestion. In recent years, sublingual/oral immunotherapies based on the continuous administration of small amounts of the allergen have been developed. However, the long periods of time needed to obtain significant desensitization and the generation of adverse effects, limit their use. In order to solve these drawbacks, strategies to induce tolerance are being studied, such as the use of either adjuvant immunotherapy in order to facilitate the reversion of the Th2 response towards Th1 or the use of monoclonal antibodies to block the main immunogenic elements.

Evaluation of nanoparticles as oral vehicles for immunotherapy against experimental peanut allergy.

The aim of this work was to evaluate the potential application of an original oral immunotherapy, based on the use of nanoparticles, against an experimentally induced peanut allergy. In this context, a roasted peanut extract, containing the main allergenic proteins, were encapsulated into poly(anhydride) nanoparticles. The resulting peanut-loaded nanoparticles (PE-NP) displayed a mean size of about 150nm and a significantly lower surface hydrophobicity than empty nanoparticles (NP). This low hydrophobicity correlated well with a higher in vitro diffusion in pig intestinal mucus than NP and an important in vivo capability to reach the intestinal epithelium and Peyer's patches. The immunotherapeutic capability of PE-NP was evaluated in a model of pre-sensitized CDI mice to peanut. After completing therapy of three doses of peanut extract, either free or encapsulated into nanoparticles, mice underwent an intraperitoneal challenge. Anaphylaxis was evaluated by means of assessment of symptom scores and mouse mast cell protease-1 levels (mMCPT-1). PE-NP treatment was associated with significant lower levels of mMCPT-1, and a significant survival rate after challenge, confirming the protective effect of this formulation against the challenge. In summary, this nanoparticle-based formulation might be a valuable strategy for peanut-specific immunotherapy.

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.