Novel particulate BCG-loaded delivery system for mucosal immunization against tuberculosis

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

It has been well described that mucosal immune responses are of major important for protection against pathogens invading the organism via mucosal surfaces, such as Mycobacterium tuberculosis. Current vaccine against tuberculosis, attenuated Mycobacterium bovis bacilli Calmette-Guérin (BCG), is commonly regarded as unsatisfactory due to its limited protection in adults against tuberculosis. The recognition that physicochemical properties, and route of administration, of vaccines influence their bioavailability and immunogenicity has led to intense research in the field of vaccine delivery. Several strategies can be employed in order to increase the immunogenicity of vaccines, namely by associating the antigens with suitable carriers and by choosing alternative vaccination routes. The aim of this thesis was to investigate the biocompatibility, cellular interaction, and immunogenicity of different types of surface modified BCG, such as BCG-loaded chitosan-alginate microparticles and BCG associated with chitosan. Polymeric nano-/microparticles have emerged as promising vaccine carriers for mucosal immunization due to their ability to improve mucoadhesion and delivery of antigens to the mucosa, while acting as adjuvants. In this context, BCG was encapsulated into polymeric microparticles composed by chitosan, chitosan and alginate, or chitosan and alginate containing linoleic acid. All particles were prepared using an optimized ionic gelation method, presenting a spherical morphology within the micrometre size range, high drug encapsulation and yield of preparation. The addition of chitosan to BCG was found to reverse its surface charge, without compromising cell viability. Studies conducted on THP-1 cells have shown that, although some formulations induced a slight decrease in cell viability, all of them were considered biocompatible. In addition, the BCG-loaded microparticles were found to be quickly internalized by human macrophages. These results highlight the potential of BCG-loaded microparticles for whole live bacterial vaccine encapsulation. Two independent in vivo experiments with BALB/c mice showed that the association of BCG with polymeric microparticles or chitosan endowed the vaccine with predominant Th1 type immunogenicity following intranasal vaccination, whereas the association of BCG with polymeric microparticles containing linoleic acid enabled a balanced Th1/Th2 profile, with a specific IgG2a/IgG1 ratio around the unity. The BCG-loaded polymeric microparticles containing linoleic acid were also found to increase the production of Th1 type cytokines, namely TNF-α, IL-2 and IFN-γ, and to promote the secretion of specific IgA in the lung. These results constituted the first quantitative analysis of the immune responses elicited in mice by BCG-loaded chitosan-alginate microparticles containing linoleic acid administered by intranasal route, highlighting the potential of this carrier in improving cellular and mucosal immune responses as pre-exposure vaccine for tuberculosis. In conclusion, polymeric microparticles proved to be a versatile platform for the delivery of attenuated vaccines, with high biocompatibility and easy surface modification, thus making them potential candidates as mucosal vaccine delivery systems. The results of this study also provide information on the adjuvant effects of the innovative association of BCG with polymeric microparticles, leading to the enhancement of cellular and mucosal immune responses following intranasal immunization, as well as on their interaction with macrophages.

Prémio Caixa Geral de Depósitos - ESTeSLIPL/CGD/2012