Elsevier

Acta Tropica

Volume 154, February 2016, Pages 73-81
Acta Tropica

A new Leishmania-specific hypothetical protein, LiHyT, used as a vaccine antigen against visceral leishmaniasis

https://doi.org/10.1016/j.actatropica.2015.11.006Get rights and content

Highlights

  • Immunoproteomic approach performed in Leishmania infantum.

  • Selection from a Leishmania-specific hypothetical protein.

  • Evaluation of its immunogenicity in BALB/c mice.

  • Protective efficacy against L. infantum infection.

  • A new candidate to compose an effective vaccine against visceral leishmaniasis.

Abstract

The present study aimed to evaluate a new Leishmania-specific hypothetical protein, LiHyT, as a vaccine candidate against VL. The immunogenicity of the recombinant protein (rLiHyT) plus saponin was evaluated in BALB/c mice. In the results, it is shown that rLiHyT plus saponin vaccinated mice produced high levels of IFN-γ, IL-12, and GM-CSF after in vitro stimulation of spleen cells using both rLiHyT and Leishmania infantum SLA. The protective efficacy was evaluated after subcutaneous challenge with stationary promastigotes of L. infantum. Immunized and infected mice, when compared to the controls, showed significant reductions in the number of parasites in the liver, spleen, bone marrow, and in the paws’ draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD4+ T cells, with a minor contribution of CD8+ T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 responses, as well as a predominance of LiHyT- and parasite-specific IgG2a isotype antibodies, were also observed. The present study showed that a new Leishmania-specific protein, when combined with a Th1-type adjuvant, presents potential to be used as a vaccine against VL.

Introduction

Leishmaniasis presents a broad spectrum of clinical manifestations and is caused by different species of protozoa belonging to the genus Leishmania. The disease exhibits a high morbidity and mortality in the world, with approximately 350 million people in 98 countries at risk of contracting the infection (WHO, 2010). Approximately 0.7–1.2 million cases of tegumentary leishmaniasis (TL), and 0.2–0.4 million cases of visceral leishmaniasis (VL) are registered annually. Visceral leishmaniasis represents an important disease worldwide, leading to nearly 50,000 deaths each year (Alvar et al., 2012). Because of its remarkable impact on global public health, VL is considered one of the six major tropical diseases, and it has gained greater importance in HIV-infected individuals as an opportunistic infection in areas where both infections are endemic (Lindoso et al., 2014). Therefore, the development of new strategies, such as a vaccine to prevent the disease, should be considered urgent.

In VL, the development of a Th1 cell-mediated immune response is considered relevant to prevention and/or cure of disease. Therefore, proteins able to stimulate the development of a Th1 response could be considered as potential vaccine candidates against VL (Fernandes et al., 2008, Chávez-Fumagalli et al., 2010, Ramírez et al., 2013). In addition, the induction of a CD4+ Th1 cells response against parasite proteins is crucial in controlling infection. Cytokines such as IFN-γ are able to induce the production of nitric oxide (NO) and other compounds by infected phagocytic cells, thereby assisting to control parasiteś multiplication (Green et al., 1990, Costa et al., 2014). On the other hand, IL-4, IL-10, IL-13, and TGF-β represent important disease promoting cytokines, leading in turn to the suppression of the Th1 response and contributing to the disease progression (Stäger et al., 2003, Wilson et al., 2005).

It has been shown that Leishmania spp. proteins that react with antibodies from VL dogs could be associated with antigenicity and protective response, representing potential diagnostic markers and/or vaccine candidates against disease (Coelho et al., 2012, Costa et al., 2012). In this context, Martins et al. (2013) evaluated an amastigote-specific Leishmania infantum hypothetical protein, which was identified in a recent immunoproteomic approach performed, have being specifically recognized by antibodies in sera of dogs suffering from symptomatic VL (Coelho et al., 2012), as a vaccine candidate against VL. This protein (LiHyp1) was used combined with saponin to immunize BALB/c mice, and this association was able to induce a LiHyp1-specific Th1 immune response and conferred protection against L. infantum infection.

Thus, the present study aimed to evaluate a new Leishmania-specific hypothetical protein, namely LiHyT, which was also characterized by the previous immunoproteomic search (Coelho et al., 2012). In this study, LiHyT was identified in the stationary-phase promastigote stage of L. infantum by antibodies in sera of dogs suffering from active symptomatic VL. After partial sequencing of one of the selected spots, an amino acid sequence from a L. braziliensis hypothetical protein (XP_001564693.1) was obtained. Using this code as probe, a new amino acid sequence of a protein with code no XP_001465138.1 (LinJ.20.0050) was rescued in the L. infantum database. After, and using the different kinetoplastid and mammals sequence databases, it was achieved that the protein was conserved between cutaneous and viscerotropic Leishmania species, and that no homologous forms were found in other Trypanosomatidae or in mammalian hosts. The purpose of the present study is to present a new candidate selected in L. infantum to be used, combined with Th1 inducing adjuvants, as a protective vaccine against VL.

Section snippets

Mice

Female BALB/c mice (8 weeks age) were obtained from the breeding facilities of the Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), and maintained under specific pathogen-free conditions. This study was approved by Committee on the Ethical Handling of Research Animals from UFMG, under the protocol number 043/2011.

Parasites

L. infantum (MHOM/BR/1970/BH46) strain was used. Parasites were grown at 24 °C in Schneider’s medium (Sigma, St.

rLiHyT induces a Th1 response characterized by increase of Type 1 cytokines and of the parasite-specific IgG2a isotype antibody

In this study, LiHyT was fused as a recombinant protein to an N-terminal 6× histidine-tag, expressed in E. coli and successfully purified by nickel affinity chromatography. The recognition of rLiHyT by L. infantum-infected mice was investigated by Western-blot analysis using a pool of sera of L. infantum chronically infected BALB/c mice (Fig. 1). Using a low range protein ladder (Invitrogen™, Life Technologies, USA) (Fig. 1A), it was possible to observe that the 36 kDa purified band protein was

Discussion

In recent decades, advances in the development of vaccines against leishmaniasis have been based on molecularly defined antigens (Gurunathan et al., 1997, Coelho et al., 2003, Iborra et al., 2008, Carrión et al., 2011, Nico et al., 2014). Although second generation vaccines are currently being tested in clinical trials, the screening of new candidates will help to further increase the prophylactic efficacy of candidates against the disease. These studies have evaluated antigens in murine

Conclusion

The results of this study indicated that a new Leishmania-specific protein, LiHyT, was able to confer protection in BALB/c mice against L. infantum challenge. Protection correlated with the induction of pro-inflammatory responses characterized by the in vitro production of IFN-γ, IL-12 and GM-CSF cytokines, beside the control of the IL-4 Th2 mediated responses and IL-10 deactivating production typically produced in non-vaccinated infected mice. These in vitro findings correlated to the presence

Conflicts of interest

The authors declare that they have no conflict of interests.

Acknowledgments

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). Also, this study was partially funded by the Spanish grant from Ministerio de Economía y Competitividad-FEDER (FISPI14/00366 from the Instituto de Salud Carlos III). MACF is a grant recipient of FAPEMIG/CAPES. EAFC is a grant recipient of CNPq.

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