Elsevier

Experimental Parasitology

Volume 153, June 2015, Pages 180-190
Experimental Parasitology

Evaluation of adjuvant activity of fractions derived from Agaricus blazei, when in association with the recombinant LiHyp1 protein, to protect against visceral leishmaniasis

https://doi.org/10.1016/j.exppara.2015.03.027Get rights and content

Highlights

  • Purification of polysaccharide-rich fractions from Agaricus blazei mushroom.

  • In vitro immune stimulation experiments using spleen cells of BALB/c mice.

  • Selection of the two best adjuvant fractions (F2 and F4).

  • Protection of LiHyp1 protein plus F2, F4 or saponin against Leishmania infantum.

  • Identification of new Th1 immune response adjuvants derived from Agaricus blazei.

Abstract

The development of effective prophylactic strategies to prevent leishmaniasis has become a high priority. No less important than the choice of an antigen, the association of an appropriate adjuvant is necessary to achieve a successful vaccination, as the majority of the tested antigens contain limited immunogenic properties, and need to be supplemented with immune response adjuvants in order to boost their immunogenicity. However, few effective adjuvants that can be used against leishmaniasis exist on the market today; therefore, it is possible to speculate that the research aiming to identify new adjuvants could be considered relevant. Recently, Agaricus blazei extracts have proved to be useful in enhancing the immune response to DNA vaccines against some diseases. This was based on the Th1 adjuvant activity of the polysaccharide-rich fractions from this mushroom. In this context, the present study evaluated purified fractions derived from Agaricus blazei as Th1 adjuvants through in vitro assays of their immune stimulation of spleen cells derived from naive BALB/c mice. Two of the tested six fractions (namely F2 and F4) were characterized as polysaccharide-rich fractions, and were able to induce high levels of IFN-γ, and low levels of IL-4 and IL-10 in the spleen cells. The efficacy of adjuvant action against L. infantum was evaluated in BALB/c mice, with these fractions being administered together with a recombinant antigen, LiHyp1, which was previously evaluated as a vaccine candidate, associated with saponin, against visceral leishmaniasis (VL). The associations between LiHyp1/F2 and LiHyp1/F4 were able to induce an in vivo Th1 response, which was primed by high levels of IFN-γ, IL-12, and GM-CSF, by low levels of IL-4 and IL-10; as well as by a predominance of IgG2a antibodies in the vaccinated animals. After infection, the immune profile was maintained, and the vaccines proved to be effective against L. infantum. The immune stimulatory effects in the BALB/c mice proved to be similar when comparing the F2 and F4 fractions with a known Th1 adjuvant (saponin), though animals vaccinated with saponin did present a slight to moderate inflammatory edema on their hind footpads. In conclusion, the F2 and F4 fractions appear to induce a Th1-type immune response and, in this context, they could be evaluated in association with other protective antigens against Leishmania, as well as in other disease models.

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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