Skip Navigation

Noninvasive Real-Time Monitoring of Liver-Stage Development of Bioluminescent Plasmodium Parasites

  1. Agnes Mwakingwe1,2,
  2. Li-Min Ting1,2,
  3. Sarah Hochman1,2,
  4. John Chen3,
  5. Photini Sinnis4 and
  6. Kami Kim1,2
  1. Departments of
  2. 1Medicine and
  3. 2Microbiology and Immunology, Albert Einstein College of Medicine, Bronx,
  4. 3Kimmel Center for Biology and Medicine, Skirball Institute for Biomolecular Medicine, New York University Medical Center, and
  5. 4Department of Medical Parasitology, New York University School of Medicine, New York, New York
  1. Reprints or correspondence: Dr. Kami Kim, Ullmann 1225, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (kami.kim{at}einstein.yu.edu)
 
Next Section

Abstract

BackgroundThe morbidity and mortality associated with malaria are heightened because of the spread of drug-resistant parasites and the lack of an effective vaccine. Plasmodium liver stages are the targets of new chemotherapeutics and vaccines, but there are limited tools available to study this stage in vivo

MethodsTo overcome this obstacle, we developed a method with which to study Plasmodium liver stages by means of bioluminescent imaging (BLI) of the rodent malaria parasite Plasmodium yoelii. We created a P. yoelii YM strain (PyLuc) that stably expresses firefly luciferase driven by a constitutive promoter

ResultsUsing BLI, we performed imaging of the Plasmodium liver stages of mice infected with PyLuc sporozoites and monitored parasite dissemination during blood-stage infection. Because PyLuc luciferase activity is proportional to the number of parasites, BLI can be used to quantify the effect of drugs on liver-stage development. Moreover, using BLI, we demonstrated that immunization with blood-stage parasites confers partial protective immunity against the development of liver stages

ConclusionsBLI is a noninvasive technique that is useful for screening potential drugs and candidate vaccines with which to combat malaria. The prospect of cross-stage protective immunity increases the number of avenues to be explored in the development of an effective vaccine against malaria

As an infected mosquito takes a blood meal, Plasmodium sporozoites are deposited in the dermis, enter the circulation, and then go through an obligatory developmental stage as exoerythrocytic forms (EEFs) in hepatocytes before initiating the symptomatic erythrocytic stage of the infection. Because infection with attenuated sporozoites that do not complete the life cycle in the liver induces protective immunity [1, 2], Plasmodium liver stages are a major focus of vaccine development [3]. Drugs that target the liver stages are important for malaria prophylaxis and eradication of malaria. One of the major obstacles to identifying new drug targets and vaccine candidates is the limited number of available tools with which to evaluate the development of liver stages in vivo

A common method of evaluating liver-stage development in vivo involves assessment of the prepatent period, or the time it takes for parasites to appear in the peripheral blood after infection with sporozoites. This is an indirect method that has been useful in assessing an all-or-none effect of a drug or vaccine on liver-stage development. However, such assessment may be complicated in cases where an intervention such as a drug candidate has effects on both EEFs and erythrocytic forms of the parasite

Other, more-direct methods used to study development of the parasite in the liver in vivo include histopathologic examination of liver sections [4], real-time quantitative reverse-transcription polymerase chain reaction (real-time qRT-PCR) [5, 6], flow cytometry of green fluorescent protein (GFP)–tagged parasites [7], and intravital imaging of GFP-tagged Plasmodium organisms [7–9]. These methods have represented significant advances, but they require killing mice to harvest livers, precluding longitudinal studies of infection. Because of the inherent biological variability of infection in individual mice, a large number of animals may be required for each experiment, to achieve statistical power. Furthermore, these methods are labor intensive and expensive. We therefore pursued a more efficient method of studying Plasmodium liver stages in vivo that would enable us to monitor infected mice longitudinally

Bioluminescent imaging (BLI) is a technique that captures light emitted by the reaction of the luciferase and its substrate. For in vivo visualization, microorganisms are engineered to express firefly luciferase, and the substrate d-luciferin is injected into the animal systemically. BLI has been successfully used to study microbial and viral pathogenesis in vivo [10, 11]. Recently, Franke-Fayard et al [12] generated a luciferase-expressing Plasmodium berghei parasite that was successfully used to study sequestration during blood-stage infection in mice. However, the authors did not image liver stages in vivo

Plasmodium yoelii is commonly used to study the efficacy of agents against liver stages, because its sporozoites infect hepatocytes efficiently and develop large numbers of liver-stage parasites [7]. P. yoelii has also been used extensively for immunology and vaccine studies, because it is thought to better mimic in experimental animals the immune response to human malarias [13]. Because even low inocula of P. yoelii sporozoites result in the release of thousands of merozoites into the blood, partial therapeutic effects of drugs are not accurately measured by analysis of the prepatent period

We hypothesized that BLI would address many of the limitations of the current methods used to study liver stages in vivo. We therefore generated a transgenic P. yoelii parasite that constitutively expresses a GFP-luciferase transgene. With this parasite, we can qualitatively and quantitatively analyze Plasmodium liver and erythrocytic stages in vivo for drug and vaccine efficacy studies

Previous SectionNext Section

Materials and Methods

Infection of mice with blood-stage parasites or sporozoitesNaive 8-week-old female BALB/c (Charles River) mice were each intravenously infected with 2×105 infected red blood cells (iRBCs) of either PyLuc or wild-type (WT) P. yoelii YM parasites; 5 mice were included in each infection group. Parasitemia was monitored by Giemsa staining of blood smears obtained on a daily basis. Parasites were harvested by intraocular bleeding of infected mice. In some cases, salivary gland sporozoites were dissected, counted, and injected intravenously. Rodent malaria infections were achieved using protocols approved by the animal use committees at the Albert Einstein College of Medicine and the New York University School of Medicine, in facilities approved by the Association for Assessment and Accreditation of Laboratory Animals

Real-time in vivo BLI of mice and dissected organs Luciferase activity was measured using an intensified charged-coupled device video camera of the In Vivo Imaging System (IVIS 100; Xenogen). WT- and PyLuc-infected animals were intravenously injected through the tail with 200 μL of d-luciferin sodium salt (Synchem OHG; catalogue number BC218) dissolved in phosphate-buffered saline (100 mg/kg of body weight). Luciferin distributed systemically for 3 min while the animals were anesthetized with the use of isofluorane. Anesthetized animals were placed in the camera chamber, and a bioluminescence signal was acquired for 5 min. Organs were dissected 3 min after administration of d-luciferin and were placed in a petri dish for imaging. Bioluminescence measurements produced by the IVIS 100 system are expressed as a pseudocolor on a gray background, with red denoting the highest intensity and blue the lowest

To quantify luminescence, we outlined a region of interest and analyzed it by use of Living Image (version 2.11; Xenogen) and Igor Pro (version 4.02A [13]; WaveMetrics) software [14]. Images of mice infected by mosquito bites are displayed with the minimal signal set at 4000 photons

Quantification of the parasite load in organs by means of real-time qRT-PCRAt 44 h after infection, liver, spleen, and lung were harvested from PyLuc-infected and uninfected mice. Total RNA was isolated using Tri-Reagent (Molecular Research Center), in accordance with the manufacturer’s protocol. Reverse transcription was performed with 1 μg of RNA and random hexamers (Invitrogen). The parasite load was quantified by qRT-PCR [6] performed using primers 5′-GGGGATTGGTTTTGACGTTTTTGCG-3′ (Py5EEFLiv) and 5′-AAGCATTAAATAAAGCGAATACATCCTTAT-3′ (Py3EEFLiv), which were specific for P. yoelii 18S ribosomal RNA [6], with the use of Power SYBR Green PCR Master Mix (Applied Biosystems). Primers specific for mouse glyceraldehyde-3-phosphate dehydrogenase (SuperArray) were used as quantitative internal controls for RNA levels. The results are expressed as the fold change comparing the parasite load in infected mice versus that in uninfected mice

Drug treatmentsA total of 75–100 PyLuc-infected mosquitoes were allowed to bite 5 anesthetized mice. To normalize infection, mice were rotated between cages, with each rotation lasting 1–2 min, for a total of 5 rounds. Infected mice were then randomly assigned to groups for drug treatment

Drugs were then dissolved. For example, to inject 12 mg/kg, we first dissolved 12 mg of the drug in 1 mL of dimethyl sulfoxide and then diluted it in 0.05% Tween 80 H2O, for a total of 10 mL. We then injected 200 μL of this solution into mice with a body weight of 20 g. Atovaquone and primaquine were administered intraperitoneally, whereas chloroquine (CQ; 120 mg/kg) was administered subcutaneously. All mice were treated once, at 1 h after infection. Untreated mice infected with either PyLuc or WT were used as control mice

Mice immunization and challengeFor immunization, Swiss Webster mice (Taconic) or BALB/c mice (Charles River) were intravenously infected with 2×104 iRBCs of P. yoelii 17XNL (Py17XNL BS; a nonlethal strain) or 2×104 iRBCs of ΔPyPNP (attenuated P. yoelii YM parasites lacking purine nucleoside phosphorylase [PNP]) [15] or by bites from mosquitoes infected with P. yoelii 17XNL (200 mosquitoes were allowed to bite 5 mice). At 4–6 weeks after infection, mice were challenged by bites from PyLuc-infected mosquitoes, as described above, and were imaged 42 h later

To determine the parasite load by use of qRT-PCR, 7-week-old female BALB/c mice (Charles River) were immunized as described above. At 55 days after the blood-stage immunizations or 44 days after immunization with sporozoites, groups of 5 mice were challenged by bites from 200 P. yoelii 17XNL–infected mosquitoes. Livers were harvested 40 h after challenge. Total RNA was isolated, and 4 μg of RNA was used for qRT-PCR

Statistical analysisGraphPad Prism software (version 5) was used for statistical analysis. Nonparametric t test analysis was used to compare groups of PyLuc-infected mice with WT-infected mice

Previous SectionNext Section

Results

Detection of PyLuc blood-stage parasitemia by BLIWe generated a clone of P. yoelii YM (WT) parasites that express a GFP-luciferase fusion protein under a constitutive promoter (PyLuc) (Figure 1, which does not appear in the print version of the article, which appears only in the electronic version of the article). These parasites behave similarly to WT parasites and are able to complete the life cycle (Figure 2, which does not appear in the print version of the articleFigure 2, which appears only in the electronic version of the article). To determine the efficiency of BLI during intraerythrocytic infection, we performed imaging of mice infected with WT and PyLuc (Figure 3A) at various levels of parasitemia. In PyLuc-infected mice, low levels of parasitemia (0.1–0.2%) (Figure 3A and data not shown) were readily detectable by BLI. Bioluminescent signal was not detected in WT-infected mice (Figure 3A)

Figure 1.
View larger version:
Figure 1.

Schematic of the green fluorescent protein (GFP)–luciferase integration strategy and verification of reporter activity of Plasmodium yoelii YM strain (PyLuc) parasites. A Use of Apa1 linearized plasmid pL0027 to stably transfect P. yoelii YM parasites by means of a single crossover integration. Kpn1 and BsiW1 were used for Southern blot analysis; predicted sizes are indicated. Primers AM1 and AM3 for diagnostic polymerase chain reaction (PCR) are noted. SB, the position of Southern blot probe. B Use of genomic DNA harvested from blood-stage parasites for integration-diagnostic PCR. Primers AM1 and AM3 amplifying the 5′ end confirmed integration by amplifying the 2.3-kb product. PCR amplification of a 1-kb band of PyADA was a positive control. Lane 1 clone AIV; lane 2 clone PyLuc; lane 3 clone DI; lane 4 clone DII; and lane 5 wild type (WT). C Digestion of 2 μg of genomic DNA harvested from blood-stage parasites by use of BsiW1 and Kpn1 for 24 h. The blot probed at the small subunit (SSU) confirms integration by the absence of the 4-kb Pyssu 2 band and the appearance of new bands: an 11.6-kb integration band and a 10.8-kb band (KpnI/BsiWI digestion removes 2 kb of pL0027 plasmid) in PyLuc. The 4.8-kb Pyssu 1 band is maintained in both WT and PyLuc strains. D Fluorescence-activated cell sorter analysis of PyLuc and WT schizonts (infected erythrocytes or infected red blood cells) harvested by 60% Accudenz/phosphate-buffered saline gradient. Compared with WT schizonts, the PyLuc GFP fluorescence peak is shifted to the right, indicating that PyLuc parasites express GFP in blood stages. E Ex vivo luciferase assay (Promega Luciferase Assay System), performed using blood samples from PyLuc-infected mice (data points from each animal are shown), showing a linear correlation between the number of infected red blood cells and the intensity of the luminescent signal. Blood from WT-infected mice and Luciferase Assay Regent (Promega) alone were used as background controls

Figure 2.
View larger version:
Figure 2.

Similar behavior of the PyLuc Plasmodium yoelii YM strain and the wild-type (WT) P. yoelii YM strain. A Six-week-old female BALB/c mice infected with 2×105 infected red blood cells of either PyLuc or WT P. yoelii YM (5 mice/group). The growth rate was monitored by calculating the percentage of infected red blood cells in Giemsa-stained blood smears. PyLuc parasites multiplied slightly slower than WT parasites. B Daily monitoring of mice after infection, until the mice died of the infection. All mice died by day 7. C PyLuc parasites and oocyst formation in mosquitoes. Female Anopheles stephensi mosquitoes were allowed to take a blood meal from PyLuc-infected mice. On day 8 after feeding, midguts were dissected and analyzed for the presence of oocysts (original magnification, ×40). PyLuc produced oocysts, as observed by phase-contrast microscopy. These oocysts express GFP, as observed by fluorescent microscopy. The arrow points to the same oocyst in phase and fluorescence

Figure 3
View larger version:
Figure 3

Correlation of luciferase activity with parasitemia. A Imaging of Plasmodium yoelii YM strain (PyLuc)–infected mice at various levels of parasitemia. Bioluminescent signal could be detected in PyLuc-infected mice with 0.2% peripheral blood parasitemia, whereas signal could not be detected in wild-type (WT)–infected mice at 91% peripheral blood parasitemia. B Detection of bioluminescent signal in dissected livers of mice bitten by PyLuc-infected mosquitoes after intravenous injection of d-luciferin, with the highest signal noted in the lungs. C Isolation of total RNA from the liver and lungs of 3 blood-stage PyLuc-infected mice with detectable bioluminescent signal overlying the lungs and from uninfected mice. Quantitative reverse-transcription polymerase chain reaction was performed using P. yoelii–specific 18S ribosomal RNA primers [6] and mouse glyceraldehyde-3-phosphate dehydrogenase primers as internal control. Analysis performed using the 2−ΔΔCt method verified a higher P. yoelii load in the lungs than in the liver, consistent with bioluminescent imaging results. The standard deviation is too small to visualize in the scale of the graph. *8000 Photons/s/cm2/steradians (Sr); †1×106 photons/s/cm2/Sr

With increasing levels of parasitemia, a stronger signal was detected overlying the thorax. We dissected lungs, heart, liver, and spleen from mice infected with either the WT or PyLuc (Figure 3B) strain, and we confirmed that the signal detected in PyLuc-infected mice originated mostly from the lungs, a finding consistent with observations in another rodent malaria model, P. berghei [12]

To compare BLI with a standard quantitative assay, we used qRT-PCR [5, 6] to detect the presence of erythrocytic-stage parasites in vivo. We harvested liver and lung tissue from PyLuc-infected mice that had detectable bioluminescent signal as well as from uninfected mice, and we measured the parasite load by use of qRT-PCR [6]. By using the 2−ΔΔCt method [16], we verified that the parasite load in the lungs and liver of PyLuc-infected mice was comparable to BLI signal intensity (Figure 3C)

Liver-stage PyLuc parasites can be detected by in vivo BLITo detect liver stages, we allowed either PyLuc- or WT- infected mosquitoes to feed on naive mice. BLI performed on day 0 and on day 1 after infection did not reveal any detectable parasite signal (data not shown); however, by 42 h after infection, bioluminescence was detected in the region corresponding to the liver in PyLuc-infected mice but not in control mice (Figure 4A)

Figure 4.
View larger version:
Figure 4.

Detection of liver stages of Plasmodium yoelii YM strain (PyLuc) 42 h after infection with sporozoites (SPZ), as achieved by bioluminescent imaging (BLI) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). A Infection of mice via mosquito bites from either PyLuc- or wild-type (WT) P. yoelii–infected female Anopheles stephensi. At 42 h after infection, WT- and PyLuc-infected mice were injected with d-luciferin and underwent imaging. Bioluminescent signal was detected in mice bitten by PyLuc-infected mosquitoes, with the highest intensity noted in the area overlying the liver, consistent with the presence of liver-stage parasites. Only background bioluminescent signal was detected in mice bitten by WT-infected mosquitoes. Bioluminescent signal intensity is indicated by the index on the far right of each figure, with red denoting the highest intensity and blue the lowest. B Dissection of organs from WT- or PyLuc-infected mice at 42 h after infection (3 min after intravenous injection of d-luciferin). Bioluminescent signal was detected in the livers of mice infected with PyLuc, consistent with the signal detected by BLI. Bioluminescent signal was not detected in organs from WT-infected mice. To compare BLI results with qRT-PCR results, RNA was isolated from liver, lung, and spleen of PyLuc-infected and uninfected mice. qRT-PCR was performed using P. yoelii 18S RNA–specific primers [6] and mouse glyceraldehyde-3-phosphate dehydrogenase primers as internal control. Results were analyzed using the 2−ΔΔCt method. *8000 Photons/s/cm2/steradians (Sr). C Determination of the sensitivity of detection of liver-stage parasites after mice were infected with 10, 100, 1000, or 10,000 PyLuc SPZ. At 42 h after infection, the mice underwent imaging, and average radiance was calculated using Living Image software (version 2.11; Xenogen). The liver signal on BLI was significantly different in mice injected with 1000 (P<.05, by t test) or 10,000 (P<.001, by t test) PyLuc SPZ, compared with mice injected with 10,000 WT SPZ. Bioluminescent signal detected in groups infected with 10 and 100 PyLuc sporozoites each could not be differentiated from background signal detected in WT-infected mice

To verify that the bioluminescent signal originated from the liver, we dissected the liver, lung, spleen, and heart 3 min after injection of d-luciferin, and we then imaged the organs. These images showed bioluminescent signal from the liver (Figure 4B), but no signal was detectable from other organs (Figure 4B). Using qRT-PCR [6], we measured the parasite load in the liver, lung, and spleen of PyLuc-infected mice at 44 h after infection. Using the 2−ΔΔCt method [16], there was a 300-fold increase in P. yoelii–specific ribosomal RNA in the livers of PyLuc-infected mice, compared with those of uninfected mice (Figure 4B)

Although bioluminescent signal in the lungs of PyLuc-infected mice had not been detected by use of BLI at 42 h after infection, we detected a significant parasite load in the lungs with the use of qRT-PCR in PyLuc-infected mice, compared with uninfected mice, at 44 h (Figure 4B). The EEFs develop within the liver for 42–44 h before releasing merozoites into the blood stream at ∼44 h [13]. Detection of parasites in the lungs at 44 h is consistent with the previously reported finding that merosomes, collections of merozoites released from the hepatocytes, are first trapped in the pulmonary capillaries [9, 17]

To determine the sensitivity of bioluminescent imaging, we intravenously infected mice with 10, 100, 1000, or 10,000 PyLuc sporozoites. As a control, we infected mice with 10,000 WT sporozoites. The mice underwent imaging 42 h later. Bioluminescent signal that was significantly different from background was detected in mice infected with 1000 or 10,000 PyLuc sporozoites (P<.05) (Figure 4C). Mice infected with 10 or 100 PyLuc sporozoites had bioluminescent signal indistinguishable from background

BLI evaluation of drug efficacy againstP. yoeliiliver stagesWe then tested whether PyLuc can be used to assess full and partial effects of drugs on liver stages. We infected mice by exposure to PyLuc-infected mosquitoes and then treated mice with: (1) 120 mg/kg CQ; (2) 12 mg/kg primaquine; (3) 60 mg/kg primaquine; (4) 0.18 mg/kg atovaquone; (5) 1.8 mg/kg atovaquone; (6) 18 mg/kg atovaquone; or (7) water with 0.05% Tween 80. Mice were treated only once. Mice underwent imaging 42 h after infection, and the signal was quantified using Living Image software. Three untreated PyLuc-infected mice had detectable liver signal with a prepatent period of 3 days (Figure 5A), whereas 1 untreated PyLuc-infected mouse had background signal in the liver and a prepatent period of 4 days. Representative images for all treatment groups are shown in Figure 5B

Figure 5.
View larger version:
Figure 5.

Use of bioluminescent imaging to analyze the effect of drugs on Plasmodium liver stages. Mice were infected by bites from mosquitoes infected with Plasmodium yoelii YM strain PyLuc. At 1 h after infection, mice were separated into groups, and each was treated with 1 dose of 12 mg/kg primaquine (PQ); 60 mg/kg PQ; 0.18 mg/kg, 1.8 mg/kg, or 18 mg/kg atovaquone (Ato); 120 mg/kg chloroquine (CQ); or 0.05% Tween (untreated). At 42 h after infection, mice underwent imaging for detection of bioluminescent signal. Untreated mice infected with wild-type (WT) parasites also underwent imaging. A Average radiance calculated using Living Image software (version 2.11; Xenogen). CQ has no effect on Plasmodium liver stages, whereas PQ and Ato have a dose-dependent effect on liver stages. B Representative bioluminescence images are also shown. Two different mice (untreated 1 and 2) from the phosphate-buffered saline–treated PyLuc group are shown. *8000 Photons/s/cm2/steradians (Sr)

CQ has been reported to have no effect on liver stages [18, 19]. The CQ-treated mice had a variable PyLuc load in the liver, but all mice developed parasitemia (Figure 5A and 5B). This variability is likely the result of biological variability in the number of sporozoites injected by mosquitoes during infection [20]. Two of 4 mice had high liver signal with a prepatent period of 3 days. In contrast, the remaining 2 mice had background signal with prepatent periods of 4 and 5 days

Primaquine is very effective on liver stages and is used for malaria prophylaxis and to prevent relapses of Plasmodium vivax and Plasmodium ovale malaria [18, 21, 22]. A dose-dependent effect of primaquine was observed; bioluminescent signal was detected in mice treated with 12 mg/kg but not in mice treated with 60 mg/kg (Figure 5). All mice treated with 12 mg/kg primaquine developed peripheral blood parasitemia. The mouse with the highest bioluminescent signal had a prepatent period of 3 days, whereas the 3 remaining mice had prepatent periods of 4 days. None of the mice treated with 60 mg/kg primaquine developed detectable parasitemia

Atovaquone has effects on both liver and blood stages [23, 24]. Bioluminescent signal in the liver was detected in groups treated with lower doses of atovaquone (1 dose of 0.18 mg/kg or 1.8 mg/kg) (Figure 5). Peripheral blood parasites were detected on day 4 in all mice that received 0.18 mg/kg atovaquone. Despite the detection of liver bioluminescence, blood-stage parasites were not detected in mice treated with 1.8 mg/kg atovaquone. Atovaquone has a long half-life [24], and at higher doses the residual drug in the bloodstream after a single dose may be sufficient to eliminate blood-stage parasites. Neither liver bioluminescence (Figure 5) nor peripheral blood parasites were detected in mice treated with 18 mg/kg atovaquone, a finding consistent with inhibition of EEF development

Immunization with blood-stage parasites may inhibit development of liver stagesPrevious studies demonstrated that immunization of humans and mice with ultralow doses of blood-stage malaria parasites, followed by drug treatment, results in protective immunity against subsequent blood-stage challenge [25, 26]. It has been assumed that this immunity was blood-stage specific. Our laboratory reported that infection with blood stages of attenuated P. yoelii ΔPyPNP YM parasites (parasites lacking PNP) conferred immunity on subsequent challenge with P. yoelii 17XNL sporozoites (nonlethal strain) [15]. We used BLI to examine whether ΔPyPNP vaccination protects against the development of parasites in the liver

We immunized a group of mice with blood stages of ΔPyPNP or Py17XNL BS or with Py17XNL sporozoites. Three weeks later, all mice had naturally cleared the infection. Four to 5 weeks after infection, mice were exposed to PyLuc-infected mosquitoes. At 42 h after challenge, 22% of mice immunized with Py17XNL BS, 20% of mice immunized with Py17XNL sporozoites, and 0% of mice immunized with ΔPyPNP were positive for hepatic bioluminescent signal (Table 1 and Figure 6A). Peripheral blood parasites were observed on day 3 after challenge in 22% of Py17XNL BS–immunized mice but were cleared by day 4 after challenge. Peripheral blood parasites were not detected in the other immunized mice. We detected liver bioluminescent signal in 80% of naive mice, with a prepatent period of 3 days noted for 100% of the naive mice (Table 1 and Figure 6A). Naive mice died 13 days after infection, whereas all immunized mice survived. These results suggest that, in addition to protection against erythrocytic stages, there is protection against the development of liver stages after immunization with blood-stage parasites

Figure 6.
View larger version:
Figure 6.

Immunization with blood-stage parasites leading to a partial protective immunity against development of liver stages after challenge with sporozoites (SPZ), as shown by bioluminescence imaging (BLI) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). A Mice challenged by bites from PyLuc-infected mosquitoes at 40–50 days after immunization. Immunization with Plasmodium yoelii 17XNL BS, P. yoelii 17XNL SPZ, and ΔPyPNP reduces the no. of liver-stage parasites to levels below the limit of detection of BLI. Liver-stage parasites could be detected in naive mice by use of BLI. B Mice immunized with P. yoelii 17XNL BS or ΔPyPNP or exposed to P. yoelii 17XNL–infected mosquitoes. At 44–55 days after immunization, all groups were challenged with bites of P. yoelii 17XNL–infected mosquitoes, and livers were harvested 40 h later. The parasite load was analyzed by qRT-PCR performed using P. yoelii 18S rRNA–specific primers [6] and mouse glyceraldehyde-3-phosphate dehydrogenase primers as internal control. The fold change comparing challenged mice with uninfected mice, as analyzed by the 2−ΔΔCt method, showed partial reduction of the parasite load in blood-stage–immunized mice and a higher reduction in mice immunized with SPZ. BS, blood stages; ΔPyPNP transgenic P. yoelii YM lacking purine nucleoside phosphorylase blood stages [15]; +, Animals with liver bioluminescence

Table 1
View larger version:
Table 1

Effects of Immunization on the Development of Liver Stages (LS) after Challenge with Plasmodium yoelii YM (PyLuc) Strain Sporozoites (SPZ), as Analyzed by Bioluminescence Imaging (BLI)

To confirm the results obtained by BLI, we immunized mice and challenged them with bites from P. yoelii 17XNL–infected mosquitoes. We killed the mice 40 h later, to determine the parasite load in the liver by use of qRT-PCR. There was an average 2-fold decrease in the development of liver stages in mice immunized with Py17XNL BS and in those immunized with ΔPyPNP and an average 10-fold decrease in mice immunized with Py17XNL sporozoites, relative to nonimmunized mice (Figure 6B). The combination of BLI and qRT-PCR results suggests that immunization with blood-stage whole parasites confers partial protection against the development of liver stages of Plasmodium

Previous SectionNext Section

Discussion

We report an efficient technique for studying liver stages of Plasmodium yoelii in vivo. Currently, the development of Plasmodium EEFs in the liver is not well understood because of a lack of tools with which to study this stage. Using BLI, we could detect EEFs 42 h after infection. Initially after invasion of hepatocytes, sporozoites do not replicate; instead, the parasite embarks on a program of cellular remodeling before initiation of proliferation (I. Coppens, personal communication). It is possible that a promoter stronger than elongation factor–1α or a promoter more active during liver-stage development might result in a detectable signal within the first 24 h of hepatic development

Bioluminescent signal from the liver was detected 42 h after administration of an inoculum of as few as 1000 sporozoites (Figure 4C). This level of sensitivity of detection is consistent with the average infectious dose when mice are infected with PyLuc via mosquito bites. In experiments where liver bioluminescent signal is detected at 42 h, we estimate that ∼1000 sporozoites are injected into each mouse when 5 mice are exposed to ∼75–100 infected mosquitoes. Each mosquito injects ∼100 sporozoites when it probes, although there is a wide range in the numbers of injected sporozoites [20]

A commonly used protocol for testing “causal” prophylaxis involves injecting mice intraperitoneally with 250,000 P. yoelii sporozoites in mosquito suspension [27, 28]. In our hands, intraperitoneal injection of either sporozoites or erythrocytic forms produces more variable infections than does infection produced by intravenous injection. However, infection by mosquito bite is simpler than intravenous inoculation, is biologically relevant, and results in efficient infection of the liver by sporozoites. Challenges with Plasmodium-infected mosquitoes are also commonly used for human trials of malaria vaccines

Despite our efforts to normalize the sporozoite dose injected by mosquitoes into each animal, some animals occasionally received higher (or lower) doses of sporozoites, which were reflected in liver bioluminescence detected 42 h later (Figure 4A and 4B). In assays where the route of infection does not affect the outcome of the experiment, intravenous or intradermal injection of known numbers of sporozoites (Figure 4C) should reduce the variability

The PyLuc strain provides a new tool for studying the efficacy of therapeutic interventions, particularly those that may target EEFs in the liver. Because of the short duration of the liver stage in rodent malarias and the efficiency of infection by P. yoelii partial effects of drugs have been difficult to document. However, as illustrated in our experiments with primaquine, the efficacy of different doses can be measured more accurately using BLI. Also, BLI is useful for monitoring the efficacy of drugs, such as atovoquone, that may be active for both causal prophylaxis (of EEFs) and suppressive prophylaxis (of blood stages)

PyLuc also enables us to evaluate the efficacy of attenuated strains to confer immunity to subsequent challenge. Research in our laboratory has shown that when mice naturally clear Py17XNL BS or ΔPyPNP blood-stage infection, they acquire protective immunity against subsequent challenge with either sporozoites or blood-stage parasites [15]. Using BLI and qRT-PCR, we show that immunization with blood-stage parasites leads to partial protection from development of liver stages after subsequent challenge with sporozoites. Both P. yoelii YM and Py17XNL were used for immunization and challenge, indicating that immunity is not strain specific

Although liver-stage protection acquired from blood-stage infection is less effective than that reported in association with immunization with attenuated sporozoites, immunization with erythrocytic stages could have a major effect if fewer merozoites are released and overall parasitemia is lower. Because clinical symptoms of malaria are the result of erythrocytic stages, any intervention that reduces parasitemia may reduce the morbidity caused by malaria. Vaccines with effects on both liver and blood stages should also facilitate rapid clearance of parasites

Cross-stage immunity was not investigated until recently, because of technical challenges. In a parallel study, Belnoue et al [29] also reported that immunization of mice with blood stages of a nonlethal strain of P. yoelii under chloroquine treatment led to immunity against both blood-stage and sporozoite challenge. The prospect of cross-stage protection provides us with more avenues to be explored in the efforts to develop a vaccine that protects against both liver and blood stages

Using BLI and PyLuc parasites, we can successfully analyze liver stages both qualitatively and quantitatively. Bioluminescent imaging is a simple and rapid technique that facilitates rapid screening of large numbers of animals and longitudinal follow-up of individual mice. These advantages decrease the number of animals required for experiments and allow for the natural biological variability seen in an individual animal during the course of malaria infection in vivo. The sensitivity of the assay may also be improved with newer generations of IVIS imaging equipment or parasites lines designed to express high levels of luciferase at the life-cycle stage of interest. These parasites have the potential to accelerate efforts to better understand Plasmodium-host interaction and development of the immune response. BLI also provides a new tool that is useful for the identification of new chemotherapy agents, particularly malaria prophylaxis candidates

Previous SectionNext Section

Acknowledgment

We thank Richard P. Novick for his thoughtful critique of the manuscript before submission for publication and for generously providing access to the In Vivo Imaging System (Xenogen)

Previous SectionNext Section

Footnotes

  • ↵Potential conflicts of interest: none reported

    Presented in part: 108th Annual Meeting of the American Society for Microbiology, Boston, Massachusetts, 1–5 June 2008; 46th Annual Meeting of the Infectious Diseases Society of America, Washington, DC, 25–28 October 2008 (abstract P-1598); and the Annual Meeting of the American Society of Tropical Medicine and Hygiene, New Orleans, Louisiana, 7–11 December 2008

    Financial support: US Army Research (grant W81XWH-05-2-0025 to K.K.) and National Institutes of Health (grant R01 AI056840 to P.S.). Initial experiments were supported by a pilot grant from the Albert Einstein College of Medicine Marion Bessin Liver Research Center, which is supported by the National Institute of Diabetes and Digestive and Kidney Diseases Core Research Center (grant P30 DK41918)

    Portions of this work were published in a thesis submitted in partial fulfillment of the requirements for a doctor of philosophy degree in the Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York (A.M.)

  • Received April 1, 2009.
  • Accepted June 10, 2009.
Previous Section
 

References

  1. ↵
    1. Nussenzweig V,
    2. Nussenzweig RS
    . Rationale for the development of an engineered sporozoite malaria vaccine. Adv Immunol45:283-334.
  2. ↵
    1. Hoffman SL,
    2. Goh LM,
    3. Luke TC,
    4. et al
    . Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J Infect Dis185:1155-64.
  3. ↵
    1. Hoffman SL,
    2. Doolan DL
    . Malaria vaccines-targeting infected hepatocytes. Nat Med6:1218-9.
  4. ↵
    1. Khan ZM,
    2. Ng C,
    3. Vanderberg JP
    . Early hepatic stages of Plasmodium berghei: release of circumsporozoite protein and host cellular inflammatory response. Infect Immun60:264-70.
  5. ↵
    1. Briones MR,
    2. Tsuji M,
    3. Nussenzweig V
    . The large difference in infectivity for mice of Plasmodium berghei and Plasmodium yoelii sporozoites cannot be correlated with their ability to enter into hepatocytes. Mol Biochem Parasitol77:7-17.
  6. ↵
    1. Bruña-Romero O,
    2. Hafalla JC,
    3. González-Aseguinolaza G,
    4. Sano G,
    5. Tsuji M
    . Detection of malaria liver-stages in mice infected through the bite of a single Anopheles mosquito using a highly sensitive real-time PCR. Int J Parasitol31:1499-502.
  7. ↵
    1. Tarun AS,
    2. Baer K,
    3. Dumpit RF,
    4. et al
    . Quantitative isolation and in vivo imaging of malaria parasite liver stages. Int J Parasitol36:1283-93.
  8. ↵
    1. Sturm A,
    2. Graewe S,
    3. Franke-Fayard B,
    4. et al
    . Alteration of the parasite plasma membrane and the parasitophorous vacuole membrane during exo-erythrocytic development of malaria parasites. Protist160:51-63.
  9. ↵
    1. Sturm A,
    2. Amino R,
    3. van de Sand C,
    4. et al
    . Manipulation of host hepatocytes by the malaria parasite for delivery into liver sinusoids. Science313:1287-90.
  10. ↵
    1. Contag CH,
    2. Contag PR,
    3. Mullins JI,
    4. Spilman SD,
    5. Stevenson DK
    . Photonic detection of bacterial pathogens in living hosts. Mol Microbiol18:593-603.
  11. ↵
    1. Luker GD,
    2. Bardill JP,
    3. Prior JL,
    4. Pica CM,
    5. Piwnica-Worms D
    . Noninvasive bioluminescence imaging of herpes simplex virus type 1 infection and therapy in living mice. J Virol76:12149-61.
  12. ↵
    1. Franke-Fayard B,
    2. Janse CJ,
    3. Cunha-Rodrigues M,
    4. et al
    . Murine malaria parasite sequestration: CD36 is the major receptor, but cerebral pathology is unlinked to sequestration. Proc Natl Acad Sci U S A102:11468-73.
  13. ↵
    1. Khan ZM,
    2. Vanderberg JP
    . Role of host cellular response in differential susceptibility of nonimmunized BALB/c mice to Plasmodium berghei and Plasmodium yoelii sporozoites. Infect Immun59:2529-34.
  14. ↵
    1. Franke-Fayard B,
    2. Waters AP,
    3. Janse CJ
    . Real-time in vivo imaging of transgenic bioluminescent blood stages of rodent malaria parasites in mice. Nat Protoc1:476-85.
  15. ↵
    1. Ting LM,
    2. Gissot M,
    3. Coppi A,
    4. Sinnis P,
    5. Kim K
    . Attenuated Plasmodium yoelii lacking purine nucleoside phosphorylase confer protective immunity. Nat Med14:954-8.
  16. ↵
    1. Livak KJ,
    2. Schmittgen TD
    . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods25:402-8.
  17. ↵
    1. Baer K,
    2. Klotz C,
    3. Kappe SH,
    4. Schnieder T,
    5. Frevert U
    . Release of hepatic Plasmodium yoelii merozoites into the pulmonary microvasculature. PLoS Pathog3:e171.
  18. ↵
    1. Belnoue E,
    2. Costa FT,
    3. Frankenberg T,
    4. et al
    . Protective T cell immunity against malaria liver stage after vaccination with live sporozoites under chloroquine treatment. J Immunol172:2487-95.
  19. ↵
    1. Most H,
    2. Montuori WA
    . Rodent systems (Plasmodium berghei–Anopheles stephensi) for screening compounds for potential causal prophylaxis. Am J Trop Med Hyg24:179-82.
  20. ↵
    1. Medica DL,
    2. Sinnis P
    . Quantitative dynamics of Plasmodium yoelii sporozoite transmission by infected anopheline mosquitoes. Infect Immun73:4363-9.
  21. ↵
    1. Bates MD,
    2. Meshnick SR,
    3. Sigler CI,
    4. Leland P,
    5. Hollingdale MR
    . In vitro effects of primaquine and primaquine metabolites on exoerythrocytic stages of Plasmodium berghei. Am J Trop Med Hyg42:532-7.
  22. ↵
    1. Boulard Y,
    2. Landau I,
    3. Miltgen F,
    4. Ellis DS,
    5. Peters W
    . The chemotherapy of rodent malaria, XXXIV. Causal prophylaxis part III: ultrastructural changes induced in exo-erythrocytic schizonts of Plasmodium yoelii by primaquine. Ann Trop Med Parasitol77:555-68.
  23. ↵
    1. Hudson AT
    . Atovaquone—a novel broad-spectrum anti-infective drug. Parasitol Today9:66-8.
  24. ↵
    1. Hudson AT,
    2. Dickins M,
    3. Ginger CD,
    4. et al
    . 566C80: a potent broad spectrum anti-infective agent with activity against malaria and opportunistic infections in AIDS patients. Drugs Exp Clin Res17:427-35.
  25. ↵
    1. Pombo DJ,
    2. Lawrence G,
    3. Hirunpetcharat C,
    4. et al
    . Immunity to malaria after administration of ultra-low doses of red cells infected with Plasmodium falciparum. Lancet360:610-7.
  26. ↵
    1. Elliott SR,
    2. Kuns RD,
    3. Good MF
    . Heterologous immunity in the absence of variant-specific antibodies after exposure to subpatent infection with blood-stage malaria. Infect Immun73:2478-85.
  27. ↵
    1. Zhang Q,
    2. Guan J,
    3. Sacci J,
    4. et al
    . Unambiguous synthesis and prophylactic antimalarial activities of imidazolidinedione derivatives. J Med Chem48:6472-81.
  28. ↵
    1. Guan J,
    2. Zhang Q,
    3. O’Neil M,
    4. et al
    . Antimalarial activities of new pyrrolo[3,2-f]quinazoline-1,3-diamine derivatives. Antimicrob Agents Chemother49:4928-33.
  29. ↵
    1. Belnoue E,
    2. Voza T,
    3. Costa FT,
    4. et al
    . Vaccination with live Plasmodium yoelii blood stage parasites under chloroquine cover induces cross-stage immunity against malaria liver stage. J Immunol181:8552-8.
| Table of Contents

This Article

  1. J Infect Dis. (2009) 200 (9): 1470-1478. doi: 10.1086/606115
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most