Abstract
In the present study, two Leishmania infantum hypothetical proteins present in the amastigote stage, LiHyp1 and LiHyp6, were combined with a promastigote protein, IgE-dependent histamine-releasing factor (HRF); to compose a polyproteins vaccine to be evaluated against L. infantum infection. Also, the antigenicity of the three proteins was analyzed, and their use for the serodiagnosis of canine visceral leishmaniasis (CVL) was evaluated. The LiHyp1, LiHyp6, and HRF DNA coding sequences were cloned in prokaryotic expression vectors and the recombinant proteins were purified. When employed in ELISA assays, all proteins were recognized by sera from visceral leishmaniasis (VL) dogs, and presented no cross-reactivity with either sera from dogs vaccinated with a Brazilian commercial vaccine, or sera of Trypanosoma cruzi-infected or Ehrlichia canis-infected animals. In addition, the antigens were not recognized by antibodies from non-infected animals living in endemic or non-endemic areas for leishmaniasis. The immunogenicity and protective efficacy of the three proteins administered in the presence of saponin, individually or in combination (composing a polyproteins vaccine), were evaluated in a VL murine model: BALB/c mice infected with L. infantum. Spleen cells from mice inoculated with the individual proteins or with the polyproteins vaccine plus saponin showed a protein-specific production of IFN-γ, IL-12, and GM-CSF after an in vitro stimulation, which was maintained after infection. These animals presented significant reductions in the parasite burden in different evaluated organs, when compared to mice inoculated with saline or saponin. The decrease in parasite burden was associated with an IL-12-dependent production of IFN-γ against parasite total extracts (produced mainly by CD4+ T cells), correlated to the induction of parasite proteins-driven NO production. Mice inoculated with the recombinant protein-based vaccines showed also high levels of parasite-specific IgG2a antibodies. The polyproteins vaccine administration induced a more pronounced Th1 response before and after challenge infection than individual vaccines, which was correlated to a higher control of parasite dissemination to internal organs.
Highlights
Visceral leishmaniasis (VL) represents an important disease in the world, leading to nearly 50,000 deaths annually [1]
It was concluded that the mixture of the three proteins presented a better performance than soluble L. infantum antigenic extract (SLA) for the serodiagnosis of Canine visceral leishmaniasis (CVL) (Table 1)
It has been showed that Leishmania spp. proteins that react with antibodies from VL dogs could be associated with antigenicity and protective responses, representing potential diagnostic markers and vaccine candidates against leishmaniasis [5,11]
Summary
Visceral leishmaniasis (VL) represents an important disease in the world, leading to nearly 50,000 deaths annually [1]. The primary choice for the treatment of disease is based on the parenteral administration of pentavalent antimonials; parasites’ increased resistance and side effects have been registered in the patients as important problems [2,3] Other drugs, such as amphotericin B and its liposomal formulations, as well as paramomycin and miltefosine, have shown encouraging results; their use is commonly related to toxicity and/or high cost [4]. Infected dogs can remain asymptomatic, and even be classified as false-negative in both clinical evaluations and serological trials performed [8] This is an important problem, since infected dogs (even asymptomatic ones) are important domestic reservoirs of parasites, and can further contribute to transmission between sand flies and humans [11]. A precise and early diagnosis of CVL is of utmost importance [12]
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