Resumen de Acellular vaccine against shigella flexneri: New expectations from new approaches
Shigellosis or bacillary dysentery is one of the leading causes of bacterial diarrhea worldwide. Despite the decreasing in the number of deaths in the last decades, shigellosis constitutes the second cause of diarrheal deaths worldwide and the third leading cause of death among children under five, with approximately 212,000 deaths per year globally, making critical the development of new vaccines against this disease. However, despite the many efforts that have been made in order to obtain a new vaccine candidate, none has succeeded to date.
Guided by the World Health Organization (WHO) recommendations, our approach is based on the use of acellular vaccines available for mucosal administration. More specifically, we have focused our research on Outer Membrane Vesicles (OMVs), due to their widely demonstrated potential for vaccination. Nevertheless, the yield of the OMV obtention method is not the optimum required to produce large batches for clinical use and, thus, the objective of this doctoral thesis was to develop an optimized OMV-obtaining process in order to achieve a safe and effective final antigenic complex.
In this work, we present two different strategies to improve the yield. On the one hand, we focused on the optimization of the process, specifically on the bacteria inactivation process, applying a heat treatment (HT) that achieved more blebbing and, at the same time, made the process safer. Furthermore, we completely characterized the obtained product, HT, which resulted to be non-cytotoxic in vitro and in vivo, improved the immunostimulant properties of naturally released OMVs and, more importantly, was able to protect mice from a pulmonary challenge with S. flexneri.
On the other hand, we obtained a genetically modified Shigella flexneri strain with a non-polar deletion in tolR gene and characterized the OMV-antigenic products. The tolR gene codifies the TolR protein, one of the components of the Tol-Pal system involved in maintaining the integrity of the outer membrane of Gram-negative bacteria. The deletion generated a more unstable membrane, that led to an increasing of blebbing. Moreover, the new strain was less virulent to mice and appeared to be more sensitive to hydrophobic chemicals and antibiotics. Thus, the combination of both HT inactivation and the use of the mutant strain was able to increase the yield of the OMV-obtaining process more than 8-times when compared to naturally released OMVs. The obtained product, HT-tolR, was completely characterized, was non-cytotoxic and studies on the antibody response showed a robust and diverse immune response in mice.
Finally, regarding to an easy, cheap and massive administration way of the OMV-antigenic complex, mucosal and intradermal routes were considered in this work. As mucosal route, the intranasal administration was evaluated, and, in order to improve it, a thermo-sensitive hydrogel formulation for vaccine delivery was also studied. These hydrogels are able to increase their viscosity when temperature rises to 37 ºC in the organism, which prolongs the OMV permanence and improves the immune response at the nasal mucosa.
In turn, for intradermal route evaluation, a new dissolving micropatch/microneedle device was synthesized as painless and self-administered vaccine delivery system against Shigella infection. Promising results were obtained, since intradermal immunization with micropatches did not show any toxicity and elicited a robust antibody response and protected mice from an experimental infection with S. flexneri.
In summary, this thesis includes new advances in the development of an acellular vaccine and proposes new administration methods for a more effective, safe and massive vaccination against shigellosis.
