Elsevier

Acta Tropica

Volume 171, July 2017, Pages 8-16
Acta Tropica

Probing the efficacy of a heterologous Leishmania/L. Viannia braziliensis recombinant enolase as a candidate vaccine to restrict the development of L. infantum in BALB/c mice

https://doi.org/10.1016/j.actatropica.2017.03.008Get rights and content

Highlights

  • Enolase protein was selected by an immunoproteomic approach in L. braziliensis..

  • The immunogenicity and protective efficacy was evaluated in a murine model.

  • A partial parasitological protection was reached against L. infantum.

  • Enolase showed to be a promising candidate to protects against visceral leishmaniasis.

Abstract

In the present study, the Leishmania braziliensis enolase protein was evaluated as a vaccine candidate against visceral leishmaniasis (VL). The DNA sequence was cloned and the recombinant protein (rEnolase) was evaluated as a vaccine, associated with saponin, as an immune adjuvant. The protective efficacy of the rEnolase plus saponin combination was investigated in BALB/c mice against Leishmania infantum infection. The results revealed that the vaccine induced higher levels of IFN-γ, IL-12, and GM-CSF when a capture ELISA and flow cytometry were performed, as well as an antileishmanial nitrite production after using in vitro stimulation with rEnolase and an antigenic Leishmania preparation. The vaccinated animals, when compared to the control groups, showed a lower parasite burden in the liver, spleen, bone marrow, and paws’ draining lymph nodes when both a limiting dilution technique and RT-PCR assay were performed. In addition, these mice showed low levels of antileishmanial IL-4, IL-10, and anti-Leishmania IgG1 isotype antibodies. Partial protection was associated with IFN-γ production, which was mainly mediated by CD4+ T cells. In conclusion, the present study’s data showed that the L. braziliensis enolase protein could be considered a vaccine candidate that offers heterologous protection against VL.

Introduction

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the Leishmania genus and is transmitted to mammalian hosts through the bite of an infected female phlebotomine sand fly (Bates et al., 2015). The disease is endemic in 98 countries, with more than 380 million people at risk (WHO, 2016). Infections with Leishmania donovani or L. (chagasi) infantum species result in clinical outcomes that range from an asymptomatic infection to fatal visceral leishmaniasis (VL) (Gardinassi et al., 2016). The treatment of disease presents some problems (Kobets et al., 2012, Singh et al., 2012), which have made it necessary to develop alternative control measures, such as vaccinations (Kaye and Aebischer, 2011, Agallou et al., 2014).

The development of infective promastigote forms is an important prerequisite for the transmission of disease. The sand fly midgut microbiome is a critical factor for Leishmania growth and differentiation to its infective state prior to parasite transmission (Kelly et al., 2017). Although the development of a successful preventive vaccine for human leishmaniasis was still not achieved, control measures to curtail vector-mediated transmission in areas of endemicity are desirable. The introduction of VL transmission in non-endemic areas has motivated intensive programs using the traditional measures of infection detection in dogs, with culling of seropositive animals and insecticide spraying of households (Costa et al., 2014, Coura-Vital et al., 2014).

In addition, studies have shown that about 12% to 20% of infected humans develop the clinical disease, with this fact normally linked to the prevalence of factors including malnutrition, poverty, among others (Gardinassi et al., 2016). However, although developing an asymptomatic infection, humans can constitute a reservoir of the parasites (Kumar and Nylén, 2012, Joshi et al., 2014). In this context, to potentially reach an optimized vaccine formulation, there is an essential need to understand what types of immune responses are operating in these asymptomatic individuals. Therefore, the development of any formulation aimed to protect against local and or systemic infection that can occur in humans remains a challenge. This fact represents an important problem and reflects the complexity of parasite-host interactions on leishmaniasis, which have evolved over decades (Reed et al., 2016).

Murine models have been used to shown that the Th1 cell-mediated immunity is important to protect against Leishmania infection (Das and Ali, 2012, Chávez-Fumagalli et al., 2010, Ramírez et al., 2013, Costa et al., 2014). The induction of CD4+ Th1 cell response against parasite antigens, based on the production of cytokines such as IFN-γ, IL-12, and GM-CSF that induce nitric oxide (NO) production by infected phagocytic cells, is necessary to control parasite replication (Green et al., 1990, Lage et al., 2015). By contrast, IL-4, IL-10, IL-13, TGF-β, among others, represent disease-promoting cytokines, leading in turn to the suppression of the Th1 response and contributing to the progression of infection (Wilson et al., 2005, Joshi and Kaur, 2014).

Enolase is an enzyme involved in glycolysis and glyconeogenesis. Besides its metabolic role, it is also related to other biological functions in distinct organisms (Pancholi, 2001). In Leishmania, enolase proved to be expressed on the cell surface (Quiñones et al., 2007) and to have a role in binding to the host’s plasminogen, thus presenting a function in the infectivity found in macrophages (Pires et al., 2014).

An important challenge for the development of an antileishmanial vaccine is the low efficacy of the tested antigens to protect against different Leishmania spp., since candidates usually offer a species-specific protection (Duarte et al., 2016). In the present study, the L. braziliensis enolase protein, which was first identified by an immunoproteomic approach by antibodies from tegumentary leishmaniasis (TL) patients (Duarte et al., 2015), was cloned and evaluated as a cross-protective antigen, associated with saponin, against L. infantum infection.

Section snippets

Mice and parasites

This study was approved by the Ethical Handling of Research Animals (protocol number 043/2011) from the Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil. BALB/c mice (8 weeks of age) 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. L. infantum (MOM/BR/1970/BH46) and L. braziliensis (MHOM/BR/1975/M2904) strains were used.

Immune response developed in the rEnolase/saponin-immunized mice before and after infection

In this study, rEnolase was cloned, expressed, purified (Fig. 1), and used as a vaccine candidate against L. infantum infection. To characterize the immune response induced by vaccination with the rEnolase plus saponin combination, spleen cells were collected 30 days after the last vaccine dose and stimulated with rEnolase or L. infantum SLA. In the results (Fig. 2A), anti-protein and anti-parasite IFN-γ levels were significantly higher in the vaccinated mice, when compared to the values found

Discussion

Although drugs are available for the treatment of leishmaniasis, they do not present an adequate efficacy, since they are toxic, and parasite resistance commonly increases. As a consequence, it is necessary to search for new therapeutics that could inhibit the Leishmania viability, while causing minimal side effects with a drug that is easily administered to patients (Chávez-Fumagalli et al., 2015). However, drug discovery is a long and costly process (Hughes et al., 2011), and the development

Conclusion

The results presented here have highlighted the potential to develop a heterologous antileishmanial vaccine using the L. braziliensis enolase protein that offers a partial protection against L. infantum infection.

Conflicts of interest

The authors hereby declare that they have no conflicts of interest.

Acknowledgements

This work was supported by grants from Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais (Edital 02/2017), Instituto Nacional de Ciência e Tecnologia em Nanobiofarmacêutica (INCT Nano-Biofar), 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). EAFC is a grant recipient of CNPq. MACF is a grant recipient of CAPES/FAPEMIG.

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