Effect of antiretroviral protease inhibitors alone, and in combination with paromomycin, on the excystation, invasion and in vitro development of Cryptosporidium parvum

Vera Hommer, Jutta Eichholz and Franz Petry*

Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University, Augustusplatz/Hochhaus, D-55101 Mainz, Germany

Received 19 December 2002; returned 7 March 2003; revised 17 April 2003; accepted 4 June 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
With the spread of the human immunodeficiency virus in the early 1980s, cryptosporidiosis was regarded as an AIDS-defining disease. As an opportunistic pathogen, the intestinal parasite Cryptosporidium parvum became an important cause of chronic diarrhoea, leading to high morbidity and mortality in immunocompromised patients. To date, no effective chemotherapy is available. With the introduction of protease inhibitors (PIs) in highly active antiretroviral therapy (HAART), the incidence of cryptosporidiosis in AIDS patients has declined substantially in western countries. We have therefore tested the effect of five PIs used in HAART on the excystation, invasion and development of the parasite in a cell culture system. The human ileocaecal adenocarcinoma cell line HCT-8 served as a host cell. None of the substances had an effect on the excystation rate, and only nelfinavir moderately, but statistically significantly, inhibited the host cell invasion over a period of 2 h. There were more pronounced inhibitory effects when PIs were present over the total time of intracellular development (48 h). Indinavir, nelfinavir and ritonavir inhibited parasite development significantly. The inhibitory effect was increased when the aminoglycoside paromomycin was combined with the PIs indinavir, ritonavir, and to a lesser extent saquinavir, compared to the PIs alone.

Keywords: highly active antiretroviral therapy, HAART, AIDS, Apicomplexa, HCT-8


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infection with the apicomplexan parasite Cryptosporidium parvum leads to self-limiting diarrhoea in immunocompetent individuals, but can develop into a severe and chronic disease in immunocompromised patients. Since the spread of human immunodeficiency virus (HIV) infections in the early 1980s, cryptosporidiosis has been regarded as an important opportunistic disease in AIDS patients. In addition, C. parvum is responsible for numerous waterborne outbreaks of diarrhoea in the general population. Although >100 substances have been tested,1,2 no effective treatment for cryptosporidiosis is currently available.

The parasite is transmitted from host to host via the oocyst stage, which is resistant to environmental factors. Once ingested, the oocyst excysts to liberate the invasive stage of the parasite, the sporozoite, which primarily infects the epithelium of the small intestine. Proteolytic enzymes have been shown to be important for the invasion of host cells in all major protozoan parasites, including Plasmodium spp., Leishmania spp., and Toxoplasma gondii.3 Several reports have also demonstrated that protease activities are associated with the excystation and host cell invasion of C. parvum. Proteases of the serine and cysteine classes have been detected in partially excysted oocysts, but only serine protease inhibitors (PIs) had an inhibitory effect on the excystation process.4 In addition, an arginine metalloaminopeptidase has been identified during in vitro excystation.5 Two different types of proteolytic enzymes were found to be associated with the surface of sporozoites. A 24 kDa metallo-dependent cysteine proteinase has been identified,6 and cysteine PIs were able to block the invasion of sporozoites in cell culture, suggesting that cysteine proteases have a role in the infection of host cells (C. Peterson, cited in ref. 3). Also, an arginine aminopeptidase has been identified as an integral membrane protein.7

With the introduction of PIs as part of the highly active antiretroviral therapy (HAART) in the treatment of HIV-1 infections, a beneficial effect was noted in AIDS patients suffering from cryptosporidiosis.8,9 Patients treated with nucleoside reverse transcriptase inhibitors alone showed a poor response in terms of clinical and microbiological duration of infection. Although it has been concluded that the effect of antiretroviral PIs on infections with C. parvum was secondary following immunological restoration (increased CD4+ cell count),8 a direct antiparasitic effect of PIs on parasite development has not yet been investigated. In this report, we tested the influence of five HAART PIs on the excystation rate, host cell invasion and in vitro development of C. parvum.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Parasites

Oocysts of C. parvum (Iowa strain) passaged in newborn calves were obtained from Patricia Mason (Pleasent Hill Farm, Troy, ID, USA). According to the supplier, the oocysts had been purified by sucrose density centrifugation, washed and resuspended in PBS. Parasites were stored at 4°C in the presence of penicillin 1000 U/mL and streptomycin 1000 µg/mL. Before the experiments, the oocysts were treated with 1:20 diluted commercial bleach (concentration 3.8% sodium hypochlorite; 5 min on ice) in order to surface sterilize the parasites, and then washed three times in PBS.

Host cell line

The ileocaecal adenocarcinoma cell line HCT-8 was purchased from the ATCC (CCL244). Cells were passaged in maintenance medium at 37°C in a 6% CO2/94% humidified air incubator.

Inhibitors

PIs were stored according to the manufacturers’ instructions. Stock solutions (10 mg/mL) were stored at –20°C. Amprenavir (Agenerase, 141W94, GI268188A; GlaxoWellcome, Stevenage, UK), nelfinavir mesylate (Viracept, AG1343; Agouron, La Jolla, CA, USA) and ritonavir (Norvir, ABT-538; Abbott, Chicago, IL, USA) were dissolved in absolute ethanol or dimethyl sulphoxide (DMSO). Saquinavir (Invirase, Ro 31–8959; Roche, Welwyn Garden City, UK) was dissolved in 50% (v/v) ethanol. Indinavir sulphate (Crixivan; MSD, Rahwey, NJ, USA) was dissolved in PBS. Paromomycin (Sigma P-9297) was dissolved in PBS at 50 mg/mL.

In vitro excystation

As ethanol inhibited excystation of oocysts at concentrations >2%, substances not soluble in water were solubilized in DMSO. The oocysts were pre-incubated for 24 h in the presence or absence of PIs at 4°C. Excystation was induced in RPMI medium plus 0.75% sodium taurocholate in the presence or absence of PIs at 37°C for 2 h. After incubation, the oocysts were fixed in 4% paraformaldehyde, in order to prevent further excystation, until counted microscopically. Excystation rates were estimated in a Neubauer chamber by differential interference contrast microscopy (Axioskop 2, Zeiss). Intact, unexcysted oocysts were counted against partially excysted oocysts and empty oocyst walls. At least three experiments were performed on different days (duplicate counts per experiment). Excystation rates are given as percentages of controls (no inhibitors added).

Cytotoxicity test

Various concentrations of the inhibitors, and appropriate solvent controls and combinations of PIs and paromomycin, were tested for inhibitory effects on the viability and proliferation of the host cell line HCT-8 in an MTS/PMS assay.10 MTS tetrazolium compound was purchased from Promega (Mannheim, Germany) and phenazine methosulphate (PMS) from Sigma (Deisenhofen, Germany). In one set of experiments, inhibitors diluted in medium were incubated for 2 h with host cells, then replaced by medium, and viability was determined after 24 h by adding the MTS/PMS reagent mix. MTS–formazan formation was recorded spectrometrically at 492 nm after 1–2 h. In a second approach, inhibitors were incubated for 48 h with the host cells, and an MTS/PMS assay was performed.

Host cell invasion and in vitro development of C. parvum in HCT-8 host cells

To test the effects of PIs on the development of C. parvum in vitro a cell culture system, originally described by Upton et al.,11 was adopted. Ninety-six-well cell culture plates (NUNCLON Surface, Nunc) were seeded with 1 x 105 HCT-8 cells per well 18 h before adding C. parvum oocysts. Cell culture maintenance medium was replaced by C. parvum growth medium, including supplements as described,11 with 1 x 105 C. parvum oocysts. The oocysts were allowed to excyst in situ. The effect of PIs on host cell invasion was tested by simultaneous incubation of parasites with the inhibitors. After 2 h at 37°C, cell cultures were washed with PBS and incubated for 48 h with growth medium. In a second set of experiments, the effect of PIs on the intracellular development of the parasite was tested. After 2 h at 37°C, the oocysts were replaced by growth medium including PIs. Cell cultures were incubated for 48 h in the presence of PIs. For each dilution of the inhibitors, 10-fold replicates were performed in parallel. Paromomycin, which had been shown to inhibit C. parvum development in vitro,12 was used as a control. All assays were performed at least three times on different days.

For visualization of parasite development in cell culture, HCT-8 cells were seeded into tissue culture chambers on microscope slides (LabTek, Nunc). Culture conditions were analogous to 96-well cell culture plates. Intracellular stages of C. parvum were detected with a monoclonal antibody (2C3).13 An Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes, Leiden, NL) was used to detect monoclonal antibody 2C3 bound to the parasite. Slides were examined under epifluorescence, and differential interference contrast (Axioskop 2 equipped with an AxioCam HRc digital camera, Zeiss).

Enzyme immunoassay (EIA)

An EIA was established for detection of parasite development. Culture plates were washed gently with PBS and fixed with methanol for 15 min. In order to reduce intrinsic alkaline phosphatase activity, cells were treated with 10% acetic acid in saline for 6 min on ice. After washing with PBS, plates were blocked with 1% BSA in PBS that included 0.05% Tween 20. An anti-mouse IgG calf intestine alkaline phosphatase conjugate (1:1000, Sigma A-3562) was used to detect bound anti-C. parvum antibody (2C3).13 Conversion of the alkaline phosphatase substrate pNPP (Sigma N-9389) was read at 405 nm spectrometrically (Anthos ELISA reader 2001; Salzburg, Austria).

Test results were corrected using the readout of the untreated, uninfected culture (background of HCT-8 cells only). The effects of PIs on the development of C. parvum were expressed as percentages of inhibition of parasite growth compared with the untreated infected control [mean of absorbance of control (100%) – mean of absorbance of samples (percentage) = inhibition rate (percentage)].

Statistical analysis

The SPSS statistics software package (version 9.1; operated on the computing facilities of the Institute of Medical Biometry, Epidemiology and Informatics, University of Mainz) was used for analysis. The individual test results (10-fold replicates) showed a Gaussian distribution. In order to compare tests performed on different days, absolute absorbance values were converted into inhibition rates (%), which are expressed as means ± S.D. Significance of inhibition was tested using a paired Student’s t-test. P < 0.05 was regarded significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inhibition of excystation

The preparations of C. parvum oocysts used in these experiments exhibited excystation rates of 85%–93%. In order to compare the influence of PIs between individual experiments, the excystation of untreated oocysts was set to 100% in each experiment, and the excystation of PI-treated oocysts was expressed as a percentage of the untreated controls. Even at a concentration of 1 mg/mL, none of the PIs showed a significant effect on the excystation of C. parvum oocysts (Figure 1). Excystation rates could not be determined microscopically in the presence of saquinavir and nelfinavir, as precipitates formed when these substances were mixed with the excystation solution.



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Figure 1. Influence of PIs on the excystation of C. parvum oocysts. The mean of the excystation rates of untreated controls was set to 100%. All substances were at 1 mg/mL final concentration in the excystation mix. Indinavir (INV) was dissolved in PBS, ritonavir (RTV) and amprenavir (AMV) in DMSO (10% final concentration). No excystation rates could be determined for nelfinavir (NFV) or saquinavir (SQV) as precipitates formed when parasites in excystation solution were mixed with these substances (*). Error bars = S.D.

 
Inhibition of host cell invasion and parasite development

As determined by the MTS/PMS assay, the substances tested showed substantial differences in cytotoxicity on HCT-8 cells. With the exception of amprenavir and indinavir, all PIs had a cytotoxic effect at concentrations >25 µg/mL (Table 1). As a result of this inhibition of growth of HCT-8 cells, testing the effect of PIs on the development of C. parvum was restricted to a limited range of concentrations. In these studies, no combination of PIs with paromomycin had a cytotoxic effect on HCT-8 cells (Table 2). To demonstrate parasite development in HCT-8 cells, LabTek chamber slide cultures were examined using immunofluorescence tests and differential interference contrast microscopy (Figure 2). Quantification of parasite stages was performed using an EIA in 96-well cell culture plates. HCT-8 cells that were mock-infected with heat-inactivated oocysts (5 min at 60°C) gave EIA values equivalent to uninfected control cultures (data not shown). The results of the 2 h coincubation of oocysts and PIs with the host cells are summarized in Figure 3. Of the PIs present during the 2 h invasion phase, indinavir (which was tolerated by HCT-8 cells even at 1000 µg/mL), nelfinavir and amprenavir showed only marginal inhibition of C. parvum development. However, with the exception of nelfinavir at 33 µg/mL, none of the inhibition rates were statistically significant (P > 0.05). When comparing nelfinavir with the untreated infected control, Student’s t-test gave a P value of 0.017 (mean 43% inhibition; 95% confidence interval: –71.67 to –14.35).


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Table 1. Influence of PIs on the growth rate of HCT-8 cells, as determined by an MTS/PMS assaya
 

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Table 2. Influence of PIs, in combination with paromomycin, on the growth rate of HCT-8 cells, as determined by an MTS/PMS assaya
 


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Figure 2. Photomicrographs of HCT-8 cell monolayers with intracellular stages of C. parvum. (a) An immunofluorescence test was performed after 48 h in culture. (b) Differential interference contrast microscopy of the same field as in (a). Scale bar = 10 µm.

 


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Figure 3. Inhibition of HCT-8 cell invasion by C. parvum in the presence of PIs. Oocysts were allowed to excyst and invade for 2 h in the presence of PIs. EIA was performed after 48 h of culture. Drug concentrations that caused statistically significant inhibition over controls (P < 0.05) are highlighted in black. INV, indinavir; NFV, nelfinavir; SQV, saquinavir; RTV, ritonavir; AMV, amprenavir; error bars = S.D.

 
The results of the inhibition of parasite development by PIs over a period of 48 h are shown in Figure 4. Indinavir (P < 0.001), nelfinavir (P = 0.005) and ritonavir (P = 0.001) led to moderate but statistically significant inhibition of parasite development at the highest concentrations tested (means: 51%, 23% and 34%, at 200, 10, and 10 µg/mL, respectively). In this experimental set-up, paromomycin led to inhibition rates of 38% and 60% (at 200 µg/mL and 1000 µg/mL, respectively. P < 0.001). For the combinations of paromomycin and individual PIs, a significant increase of inhibition of parasite growth over paromomycin alone was recorded with indinavir, ritonavir and saquinavir (Figure 5). The combination of paromomycin 200 µg/mL plus indinavir 200 µg/mL resulted in 82% inhibition, cultures in the presence of paromomycin 1000 µg/mL plus indinavir 200 µg/mL were inhibited by 94%. The combination of paromomycin 200 µg/mL plus ritonavir 10 µg/mL resulted in 78% inhibition, cultures in the presence of paromomycin 1000 µg/mL plus ritonavir 10 µg/mL were inhibited by 88%. For saquinavir, only the combination of paromomycin 200 µg/mL plus saquinavir 10 µg/mL was statistically significant (P = 0.007) over paromomycin alone and resulted in 56% inhibition.



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Figure 4. Inhibition of parasite development in HCT-8 cell cultures. After 2 h of oocyst excystation and host cell invasion, cell layers were washed and incubated in medium in the presence of PIs for 48 h. Paromomycin (PARO) at 200 and 1000 µg/mL served as positive inhibitory controls. Drug concentrations that caused statistically significant inhibition over controls (P < 0.05) are highlighted in black. Abbreviations as in Figure 3. Error bars = S.D.

 


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Figure 5. Inhibition of parasite development.(a) Combinations of PIs with paromomycin (PARO; 200 µg/mL). (b) Combinations of PIs with paromomycin (1000 µg/mL). Drug concentrations that caused statistically significant inhibition over paromomycin controls (P < 0.05) are highlighted in black. Abbreviations as in Figure 3. Error bars = S.D.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The target of the PIs used in HAART is the HIV-1 protease, which is a member of the aspartic acid protease family.14 Proteases of this class have not been described functionally in C. parvum. However, in a random survey of the C. parvum genome, a genomic survey sequence (clone CpGR147A/B) was identified, which codes for an aspartic proteinase similar to a protein in Eimeria acervulina.15

In contrast to C. parvum, several secretory aspartyl proteases (SAPs) have been well characterized in Candida albicans. SAPs enable the binding of C. albicans to mucosal surfaces and can degrade host tissue, a prerequisite for dissemination of the disease.16 Of four HAART PIs tested, saquinavir was inhibitory to 100% of Candida strains at a concentration of 800 µg/mL in a well-diffusion test.17 Pneumocystis carinii, another important opportunistic pathogen in AIDS patients, was shown to be susceptible to PIs used in HAART in an in vitro model.18 The growth of P. carinii in human embryonic lung (HEL 299) cell cultures was inhibited up to ~60% in the presence of PIs at concentrations ~60 ng/mL (saquinavir).

Only a limited range of concentrations could be tested in the excystation and in vitro development assay of C. parvum, owing to solubility characteristics and the cytotoxicity of most of the compounds in our in vitro system. The therapeutic daily doses of PIs used in HAART reach gram amounts. Local concentrations in the intestine, the site of Cryptosporidium infection, are therefore in the mg/mL range. Such high values could only be reached with indinavir in our assays, due to its comparably low cytotoxic effect on HCT-8 cells and its solubility in aqueous solutions. HIV-1 PIs produce gastrointestinal side effects, some of which may be the result of cytotoxic effects on the enterocytes, reflecting our in vitro observation with HCT-8 cells. All HIV-1 PIs are metabolized by cytochrome P450 isoenzyme 3A4 (CYP3A4). In addition, ritonavir in particular was shown to be not only a substrate of CYP3A4, but also a potent inhibitor leading to a decrease of conversion of other substrates of this enzyme, such as other HIV-1 PIs and certain macrolide antibiotics.19 As a consequence, plasma levels of these drugs are elevated. However, no similar interaction with aminoglycoside antibiotics, such as paromomycin, has been described that could possibly explain our in vitro results, or the clinical outcome of cryptosporidiosis of AIDS patients receiving HIV-1 PIs plus paromomycin.

The underlying mode of action of PIs used in HAART on C. parvum in AIDS patients is most likely multifactorial. First, a recovery of the CD4 cell counts due to a lowering of the viral load may explain the control of cryptosporidiosis. However, the absolute number of CD4 cells appears not to be important, but rather the stage of activation and the site of occurrence in the body. The number of CD4 cells in the intestinal mucosa increases faster and to higher levels than in blood.20 Second, ritonavir has been shown to increase the secretion of type 1 cytokines, such as interferon-{gamma} and interleukin-2.21 Interferon-{gamma} plays a crucial role in the host defence against Cryptosporidium infection.22 In the in vitro cultures shown here, we found that the inhibitory action of PIs on C. parvum was limited. The inhibitory concentrations lay between those shown for C. albicans and P. carinii. The inhibitory potential of indinavir, ritonavir and to a lesser extent, saquinavir, could be augmented by co-incubation with the aminoglycoside paromomycin. There has been controversy concerning the efficacy of paromomycin in the treatment of cryptosporidiosis.2326 Whereas Hewitt et al.23 have questioned the anti-cryptosporidial effect of this antimicrobial agent, clinical studies on a limited number of patients have shown that the combination of HAART including PIs with paromomycin has led to parasite clearance in all cases, independent of the low CD4 count of the patients.27 Further, when the results of Schmidt et al.20 are reviewed carefully they show that a combination of PIs and paromomycin over a period of 4 weeks resulted in a patient with late-stage AIDS recovering from cryptosporidiosis.

The data presented here suggest a direct action of indinavir, nelfinavir and ritonavir on C. parvum. Furthermore, an additive inhibitory effect of indinavir, ritonavir and saquinavir used in HAART in combination with paromomycin was seen, which supports the clinical and microbiological findings in patients where these combinations are used.


    Acknowledgements
 
The data presented are part of the doctoral thesis of J.E. and have been presented at the Joint Annual Meeting of the German and Dutch Societies for Parasitology, 20–23 March 2002.

We thank Dr Vincent McDonald for monoclonal anti-C. parvum antibody 2C3 and Inka Kneib for excellent technical assistance. We acknowledge the advice on statistical analysis of the data by Professor Gerhard Hommel, Institute of Medical Biometry, Epidemiology and Informatics, University of Mainz.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 490 project C5). PIs were kindly supplied by Abbott (Norvir), Agouron (Viracept), GlaxoWellcome (Agenerase), MSD (Crixivan), and Roche (Invirase).


    Footnotes
 
* Corresponding author. Tel: +49-6131-393-3139; Fax: +49-6131-393-3439; E-mail: fpetry{at}mail.uni-mainz.de Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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