Infrared fluorescent imaging as a potent tool for in vitro, ex vivo and in vivo models of visceral leishmaniasis.

Estefanía Calvo-Álvarez,Yolanda Pérez-Pertejo,José Miguel Escudero-Martínez,Manuel Fresno,Ana Tejería,Carmen Punzón,Rosa M Reguera,Raquel Álvarez-Velilla,Kostantinos Stamatakis,Rafael Balaña-Fouce

doi:10.1371/journal.pntd.0003666

Abstract

BackgroundVisceral leishmaniasis (VL) is hypoendemic in the Mediterranean region, where it is caused by the protozoan Leishmania infantum. An effective vaccine for humans is not yet available and the severe side-effects of the drugs in clinical use, linked to the parenteral administration route of most of them, are significant concerns of the current leishmanicidal medicines. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.Methodology/Principal findingsWe generated two infrared fluorescent L. infantum strains, which stably overexpress the IFP 1.4 and iRFP reporter genes and performed comparative studies of their biophotonic properties at both promastigote and amastigote stages. To improve the fluorescence emission of the selected reporter in intracellular amastigotes, we engineered distinct constructs by introducing regulatory sequences of differentially-expressed genes (A2, AMASTIN and HSP70 II). The final strain that carries the iRFP gene under the control of the L. infantum HSP70 II downstream region (DSR), was employed to perform a phenotypic screening of a collection of small molecules by using ex vivo splenocytes from infrared-infected BALB/c mice. In order to further investigate the usefulness of this infrared strain, we monitored an in vivo infection by imaging BALB/c mice in a time-course study of 20 weeks.Conclusions/SignificanceThe near-infrared fluorescent L. infantum strain represents an important step forward in bioimaging research of VL, providing a robust model of phenotypic screening suitable for HTS of small molecule collections in the mammalian parasite stage. Additionally, HSP70 II+L. infantum strain permitted for the first time to monitor an in vivo infection of VL. This finding accelerates the possibility of testing new drugs in preclinical in vivo studies, thus supporting the urgent and challenging drug discovery program against this parasitic disease.

Highlights

Neglected Tropical Diseases (NTDs) are severe scourges that affect the less protected layer of the poorest population of low-income countries [1, 2]
In vivo non-invasive imaging of the visceral infection in BALB/c mice was achieved for the first time by using transgenic fluorescent parasites
These findings open up the possibility of testing vast amounts of potential compounds and allow in vivo screening of drug candidates against this severe parasitic disease in an attempt to speed up the vital drug discovery program

Summary

Introduction

Neglected Tropical Diseases (NTDs) are severe scourges that affect the less protected layer of the poorest population of low-income countries [1, 2] Their neglected consideration implies a poor attention by most of the actors involved in their eradication including Pharma Industries, which are more concerned about those diseases affecting people from more developed countries [3]. No effective vaccine candidates, either prophylactic or preventive, are under clinical trials [7], the treatment is still mainly based on old-fashioned antimony derivatives and the administration route of these drugs is parenteral To overcome these gaps, several Big Pharma companies have made available to academic researchers and supranational institutions myriads of small molecules to be tested on Leishmania on recently developed High Throughput target-based and target-free Screenings platforms (HTS) [8, 9]. New drugs are desperately needed to treat VL and phenotype-based High Throughput Screenings (HTS) appear to be suitable to achieve this goal in the coming years.

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