Cross-protective efficacy of Leishmania infantum LiHyD protein against tegumentary leishmaniasis caused by Leishmania major and Leishmania braziliensis species
Graphical abstract
Introduction
Leishmaniasis comprises a group of diseases ranging from cutaneous to visceral forms. Although tegumentary leishmaniasis (TL) is not a fatal disease, it is endemic in more than 70 countries and 90% of the cases are registered in Afghanistan, Algeria, Brazil, Pakistan, Peru, Saudi Arabia and Syria (Desjeux, 2004, Alvar et al., 2012). Different species of the parasites can act as causative agents of TL (Grimaldi and Tesh, 1993, Reithinger et al., 2007). In Brazil, it is most commonly caused by Leishmania braziliensis species, whereas Leishmania major and Leishmania tropica have been associated with disease in the Old World (Desjeux, 2004). The disease control has been difficult due to the natural features of reservoir and vectors, making the elimination of both components hard to obtain. Also, therapeutic measures are ineffective and the development of a vaccine would be the most effective measure to eliminate or, at least, control the disease (Marzochi and Marzochi, 1994, Silveira et al., 2004).
Most strains of mice infected by L. major develop small cutaneous lesions that spontaneously resolve over several weeks (Howard et al., 1980, DeTolla et al., 1981). On the other hand, certain mice strains, such as the BALB/c, develop severe and non-healing infections that visceralize to internal organs and can be fatal (Djoko-Tamnou et al., 1981, Li et al., 1997). The development of a healing versus non-healing infection is determined by immune responsé type that usually develops after parasite inoculation. Healing infections are characterized by development of a Th1 type response mediated mainly by CD4+ cells producing the macrophage-activating cytokine, IFN-γ. On the other hand, IL-4 and IL-10 produced by Th2 cells predominate in susceptible animals (Scott et al., 1988, Heinzel et al., 1991).
An additional factor which might potentially influence the course of disease and/or resistance of the animals is related to the parasiteś infectivity. In this context, L. braziliensis species is the causative agent of cutaneous and mucosal leishmaniasis, and the latter is a severe and disfiguring form of TL in humans (David and Craft, 2009). There are studies showing that BALB/c mice are partially resistant to infection with this Leishmania species: they do not develop severe lesions and usually cure the infection developing a Th1/Th2 mixed response (Childs et al., 1984, Dekrey et al., 1998, Oliveira et al., 2004). However, others have shown that the outcome of the infection of BALB/c mice by L. braziliensis can be completely changed by treating them with previous injections of Leishmania amazonensis amastigote extracts, when the animals thus become susceptible to the disease (Araújo et al., 2014, Silva et al., 2015). In addition, it was also observed that pre-isolated parasites from lesions of chronically-infected BALB/c mice were more infective than those that were in vitro cultured for long periods of time, as was observed in the L. amazonensis species (Magalhães et al., 2014).
It is postulated that to be an effective vaccine candidate against leishmaniasis an antigen should be shared by different parasite species, immunogenic in all or in the most of them, present few polymorphisms, and not present in other Trypanosomatidae (Coelho et al., 2012, Fernandes et al., 2012). However, the most of studies have worked with Leishmania proteins also expressed in other Leishmania-related parasites (Modabber, 2010, Zanin et al., 2007, Todolí et al., 2012). In this context, Leishmania-specific proteins could be considered as interesting targets for constructing effective vaccines against disease.
We have designed this study to evaluate a new vaccine candidate, a Leishmania-specific hypothetical protein, namely LiHyD (LinJ.33.3150). This protein is conserved between different Leishmania species including those causing TL, with identity values of 80% and 56% with respect L. major (LmjF.33.2990) and L. braziliensis (LbrM.33.3270) homologues, respectively. Besides of this, LiHyD putative T cell epitopes evaluated by bioinformatics tools are located in regions conserved among the different parasite species. In this context, the effects of the administration of this protein obtained as a recombinant product (rLiHyD) in BALB/c mice (combined with saponin), a formulation previously showed to be protective against Leishmania infantum (Lage et al., 2015), was evaluated as a protective vaccine against L. major or L. braziliensis challenge infection. The vaccination using rLiHyD/saponin was able to mount a Th1 response before and after infection, which was based on rLiHyD- and parasite-specific IFN-γ production mainly by CD4+ T cells, and was able to reduce the parasite burden in the infected and vaccinated animals. In this context, the present study showed a new candidate to compose a pan-Leishmania vaccine to protect against parasite species causing TL.
Section snippets
Ethics statement and animals
The Committee on the Ethical Handling of Research Animals from Federal University of Minas Gerais (UFMG) approved this study with the protocol number 043/2011. Female BALB/c mice (8 weeks old) were obtained from the breeding facilities of the Department of Biochemistry and Immunology, Institute of Biological Sciences, UFMG; and were maintained under specific pathogen-free conditions.
Parasites
L. major (MHOM/IL/1980/Friedlin) and L. braziliensis (MHOM/BR/1975/M2903) were used. Parasites were grown at 24 °C
rLiHyD is recognized by antibodies in sera of L. major or L. braziliensis-chronically infected mice
In this study, a Leishmania hypothetical protein, which was firstly identified as protective against L. infantum, was evaluated in immunoblotting assays by its reaction with antibodies in sera of L. major or L. braziliensis-infected BALB/c mice (Fig. 1). In the results, using a Ladder standard as a marker (lane A), it was shown that whereas the recombinant protein was not reactive with antibodies in sera of non-infected mice (lane B), it was specifically recognized by antibodies in sera of L.
Discussion
The measures currently available to control the spread of tropical parasitic diseases are mainly based on therapeutic interventions such as drug-treatment programs and in the vector control, which have shown unsatisfactory results and reinforced the need for prophylactic vaccination (Sacks, 2014). Studies have shown that protection against leishmaniasis is mainly based on the development of a parasite-specific Th1 immune response, mainly linked to IFN-γ secretion (Scharton and Scott, 1993,
Conclusion
The present study investigated if a Leishmania-specific hypothetical protein, which was firstly identified and evaluated as protective against L. infantum, could cross-protect BALB/c mice against two parasite species causing TL. Taken together, our data indicate that LiHyD, when administered in a recombinant format associated with a Th1 adjuvant, was able to induce a partial cross-protection against challenges using L. major and L. braziliensis. In both cases, the protective immunity was
Conflicts of interest
The authors hereby declare that they have no conflicts of interest.
Acknowledgments
The authors would like to thank to Dr. Manuel Soto (Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain) for his scientific and technical support. This work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-NanoBiofar), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014), and CNPq (APQ-482976/2012-8, APQ-488237/2013-0, and APQ-467640/2014-9). MACF is a grant
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