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 30min 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.

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 F4E. coli (1.2 x 10(8)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 challen

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.

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-Delta tolR). The S. flexneri Delta tolR 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 Delta tolR 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-Delta tolR 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-Delta tolR demonstrated a robust and diverse response in mice. Considering these promising results, HT-Delta toIR antigenic extract appears as a new potential vaccine candidate to face shigellosis.