Evaluation of adjuvant activity of fractions derived from Agaricus blazei, when in association with the recombinant LiHyp1 protein, to protect against visceral leishmaniasis
Graphical Abstract
Introduction
Visceral leishmaniasis (VL) is mainly caused by two species of parasites, the anthroponotic Leishmania donovani and the zoonotic L. infantum, for which a variety of canids serve as animal reservoirs (Ready, 2014). The incidence of VL has been estimated to be around 500,000 cases per year, with over 50,000 deaths worldwide. Approximately 90% of the cases are registered in Bangladesh, Brazil, Ethiopia, India, Nepal, and Sudan (Alvar et al., 2012). Additionally, in the nearly 98 countries that have reported the disease; 35 of them have already registered cases of VL and HIV co-infection (World Health Organization, 2010).
The treatment of the disease is still based on the administration of pentavalent antimonial compounds; however, several side effects registered in the patients, as well as the increased parasite resistance to the drugs, have also caused serious problems (Croft, Coombs, 2003, Minodier, Parola, 2010). Therefore, the development of new strategies to prevent leishmaniasis has become a high-priority (Costa et al, 2011, Lee et al, 2012).
The evidence of life-long immunity has inspired the development of vaccination protocols against the disease, but few have progressed beyond the experimental stage (Chávez-Fumagalli et al, 2010, Coelho et al, 2003, Costa et al, 2014, Ramírez et al, 2013, Ramírez et al, 2014). In these studies, the type-1 cell-mediated immunity has proven to be important as a protective response against leishmaniasis. This response commonly depends on an IL-12-driven response, leading to an IFN-γ and IL-2 production. Substantial up regulation of inducible NO synthase by IFN-γ generates NO from splenic and liver cells, thereby controlling parasite replication in these organs (Carrión et al, 2006, Oliveira et al, 2011). In contrast, IL-4, IL-10, IL-13, and TGF-β are major disease promoting cytokines, in turn leading to the suppression of the Th1 response (Afonso, Scott, 1993, Martins et al, 2013). Thus, vaccines capable of inducing the development of a Th1 immune response, primed by the production of cytokines, such as IFN-γ and IL-12, and associated with low levels of IL-4 and IL-10, could be considered promising candidates to protect against the Leishmania sp. infection (Costa et al, 2014, Fernandes et al, 2012).
Adjuvants of the immune response are defined as products capable of enhancing and/or changing an antigen-specific immune response. Biotechnological advances have enabled modern vaccines to be based on the use of rationally designed recombinant antigens associated with purified compounds as adjuvants, which present a satisfactory safety profile (Reed et al., 2013). Adjuvants can be used for multiple purposes, such as to enhance the immunogenicity of weak antigens, provide antigen-dose sparing to accelerate the immune response, reduce the need for several immunizations, and/or increase the duration of protection (Petrovsky and Aguilar, 2004). These products can be classified as immune stimulatory molecules, such as toll-like receptor ligands and saponins, or delivery systems, such as aluminum salts, emulsions, and lipid nanosystems (Fox et al., 2013).
The use of carbohydrates with adjuvant action is based on their binding to innate immune receptors (Petrovsky and Cooper, 2011). A number of carbohydrate compounds extracted from plants (Rajput et al., 2007), bacteria (Koike et al., 1998), and yeast (Pillemer and Ecker, 1941) have emerged as promising vaccine adjuvant candidates. Their protective effects induced in immunotherapy have also been proven to act against L. amazonensis (Yatawara et al., 2009), L. donovani (Cook et al., 1982), and L. major (Al Tuwaijri et al., 1987). In addition, carbohydrates' adjuvant activity against L. donovani in a golden hamster model has been registered (Cook et al, 1982, Obaid et al, 1989). In parallel to the use of carbohydrates, the employ of protein-bound polysaccharides has also been evaluated in a wide range of biological applications (Maehara et al, 2012, Ooi, Liu, 2000), and recent studies have shown their use as immune adjuvants (Engel et al., 2013).
Recently, our group demonstrated the in vitro (Valadares et al., 2011) and in vivo antileishmanial activity of Agaricus blazei against L. amazonensis and L. infantum (Valadares et al, 2012a, Valadares et al, 2012b, respectively). Compounds, such as β-glucans, tannins, glycoproteins, and polysaccharides were characterized in this mushroom, which may well be responsible for the immunological activity of the fungus (Bernardshaw et al, 2005, Gonçalves et al, 2012, Valadares et al, 2012a). Taking this into account, in the present study, six polysaccharide-rich fractions were purified from Agaricus blazei, and evaluated in in vitro experiments as inducers of the Th1 immune response. The adjuvant fractions that presented the best results were used in association with a recombinant antigen of L. infantum, rLiHyp1 (Martins et al., 2013); and the subsequent vaccine was evaluated as regards the protection of BALB/c mice against L. infantum, using saponin as a control.
Section snippets
Ethics statement
Experiments were performed in compliance with the National Guidelines, as set forth by the Institutional Animal Care (Law number 11.794, 2008), and the Committee on the Ethical Handling of Research Animals from the Federal University of Minas Gerais (UFMG), who approved this study under protocol number 067/2013.
Mice and parasites
Female BALB/c mice (8 weeks of age) were obtained from the breeding facilities of the Department of Biochemistry and Immunology, Institute of Biological Sciences (ICB), UFMG; and were
Chemical characterization of the purified fractions from Agaricus blazei and the in vitro selection of best adjuvant fractions
To select the polysaccharide-rich fractions derived from Agaricus blazei in order to perform the in vitro immune stimulation experiments, the polysaccharide, protein, and phenolic compound contents were determined, and the results are showed in Fig. 1. The total extract of Agaricus blazei (ABP) was used as a positive control for the presence of these compounds. In evaluating the protein content, only the F5 fraction presented high levels, whereas the other fractions did not present significant
Discussion
Visceral leishmaniasis is one of the main neglected tropical parasite diseases, presenting a high mortality, ranking only behind malaria in the number of victims (Alvar et al., 2012). As the protection against re-infection with L. major in murine models is possible, efforts have been performed to develop a protective vaccine; however, to date, no effective vaccine has been created (Fernandes et al., 2012). An important advantage in using vaccines based on attenuated pathogens, inactivated
Acknowledgments
This work was supported by grants from Pró-Reitoria de Pesquisa from UFMG (Edital 01/2014), Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, RHAE-456287/2012-4, APQ-482976/2012-8 and APQ-488237/2013-0). MACF is a grant recipient of FAPEMIG/CAPES. EAFC and MNM are grant recipient of CNPq.
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