A chronic bioluminescent model of experimental visceral leishmaniasis for accelerating drug discovery

Abstract

Background: Visceral leishmaniasis is a neglected parasitic disease with no vaccine available and its pharmacological treatment is reduced to a limited number of unsafe drugs. The scarce readiness of new antileishmanial drugs is even more alarming when relapses appear or the occurrence of hard-to-treat resistant strains is detected. In addition, there is a gap between the initial and late stages of drug development, which greatly delays the selection of leads for subsequent studies.

Methodology/principal findings: In order to address these issues, we have generated a red-shifted luminescent Leishmania infantum strain that enables long-term monitoring of parasite burden in individual animals with an in vivo limit of detection of 106 intracellular amastigotes 48 h postinfection. For this purpose, we have injected intravenously different infective doses (104-5x108) of metacyclic parasites in susceptible mouse models and the disease was monitored from initial times to 21 weeks postinfection. The emission of light from the target organs demonstrated the sequential parasite colonization of liver, spleen and bone marrow. When miltefosine was used as proof-of-concept, spleen weight parasite burden and bioluminescence values decreased significantly.

Conclusions: In vivo bioimaging using a red-shifted modified Leishmania infantum strain allows the appraisal of acute and chronic stage of infection, being a powerful tool for accelerating drug development against visceral leishmaniasis during both stages and helping to bridge the gap between early discovery process and subsequent drug development.

Fig 1. Generation of a L. infantum stably expressing PpyRE9h red-shifted luciferase.

A) Scheme of the structure of the 18S rRNA locus on wild type and planned integration of PpyRE9h gene. Key: utr1: non-translated region of aprt gene; utr2: 1.4 kb intergenic region from cam operon; and utr3: UTR of dhfr-ts gene; PAC; puromycin resistance cassette. B) PCR confirmation of successful integration of the reporter cassette. Primers 630/637 and 644/645 together confirm the correct integration of the reporter cassette into genome sequences. (see Table 1 for sequences). C) Growth rate of wild-type (black circle) and stably-modified promastigotes PpyRE9h+L.infantum (white circle). Parasites were counted using a Coulter counter. D) In vitro luciferase activity assay of diluted lysates from wild-type (WT) and PpyRE9h+L.infantum promastigotes after PCR confirmation. E) Minimal metacyclic promastigote and infective amastigote number detectable by the IVIS camera. Parasites were loaded in 96-well plates, D-luciferin added and the BLI signal was detected in the IVIS camera. Grey lines indicate detection thresholds determined as the mean (solid line) and mean +2SDs (dashed line) of background luminescence of wells with PBS free-parasites. F) Microscopic image of intracellular PpyRE9h+L.infantum amastigotes infecting PMA-differentiated THP-1 cell line. Bioluminescent amastigotes stained with anti-luciferase IgG (αLuc, red) and Hoechst 33342 DNA stain (H33342, cyan).

Fig 2. Evaluation of L. infantum infection in Balb/c mice by in vivo BLI.

A) Representative images of Balb/c mice infected via IV with 5x10⁴—5x10⁶ metacyclic PpyRE9h luciferase-expressing promastigotes. Pseudocolour heat-maps indicate intensity of bioluminescence from low (blue) to high (red). All images use the same log10 scale heat-map, minimum and maximum radiance values are indicated. Animals at 9 and 12 weeks postinfection are in lateral position where most of the BLI signal was detected B) Quantification of whole animal total bioluminescence for mice in the experiment illustrated in A) at 48 h postinfection. C) Time-appraisal of Balb/c mice after IV injection with 5x10⁸ red-shifted bioluminescent parasites (PpyRE9h+L.infantum) in ventral and lateral positions. Quantification of ventral and lateral bioluminescence from the experiment represented by the images in C). D-E) Correlation between in vivo bioluminescence values and parasite burdens in the liver and the spleen. Bioluminescence was measured in vivo in ROIs around liver and spleen and parasite numbers were quantified by limiting dilution assay after animals were sacrificed. Grey lines indicate detection thresholds determined as the mean (solid line) and mean +2SDs (dashed line) of background luminescence of control uninfected animals.

Fig 3. Chronic L. infantum infection in space and time.

A) Representative ventral and lateral view images of Balb/c mice taken at sequential time points over the course of 14 weeks after IV infection with 5 x 10⁸ PpyRE9h luciferase-expressing L.infantum metacyclic promastigotes (representative of n = 30). In the images corresponding to 14 wpi only lateral views are shown because most of BLI signal was detected in this position. Heat-maps are on log10 scales indicate intensity of bioluminescence from low (blue) to high (red); the minimum and maximum radiances for the pseudocolour scale are indicated. B) Animals (n = 15) were treated with 40 mg/kg/d miltefosine via oral for 5 days. Treated and untreated animals were photographed, sacrificed and the spleen and liver imaged at 48 h, 1 week and 6 weeks after the end of miltefosine treatment (15, 16 and 21 wpi). Quantification of lateral bioluminescence for mice shown in B. The red asterisk indicates the start of miltefosine treatment. In vivo radiance from untreated (black circle) and treated (white circle) animals is represented. Black asterisks indicate P-values for t-student test (B,D,E). Comparisons between miltefosine treated groups and untreated control groups (*P < 0.05; **P<0.01; ***P<0.001). Grey line indicates detection thresholds determined as the mean (solid line) and mean +2SDs (dashed line) of background luminescence of control uninfected animals. C) Ex vivo imaging (spleen and liver) in untreated and treated animals at 48h, 1w and 6 w after the end of the treatment (BLI signal results from the D-luciferin injected ex vivo). D) Spleen weights from untreated (black) and treated (white) animals at 48h, 1w and 6 w after the end of the treatment. Each point is the mean ± SD, n = 5 per group. E) Ex vivo bioluminescence signal from spleens and livers obtained from untreated (black symbols) and treated animals (white symbols) at 48 h (circle), 1w (square) and 6w (triangle) after the end of miltefosine treatment. F) Parasite burdens in untreated and treated mice determined by limited dilution assay on livers and spleens.