Research Article - (2011) Volume 2, Issue 5
Crude preparations of malaria parasite proteins from the hydrophobic and hydrophilic phases were made by Triton X-100 temperature-induced phase separation procedures. Cysteine protease was purified from the hydrophobic phase of the parasite protein preparation. Groups of BALB/c mice were immunized intraperitoneally with the purified cysteine protease and crude preparations of the parasite proteins respectively. Priming and booster immunizations were administered on days 0, 14 and 21 prior to lethal parasite challenge on day 30. The course of infection was monitored by microscopic Giemsa-stained, thin blood smears. Protection was conferred at varying thresholds in the groups of mice immunized. This was shown by a week delay in the onset of parasitemia and red blood cell invasion by parasites in the group of mice immunized with the purified cysteine protease. Pack Cell Volume, parasite burden and the mean survival time of mice in days post-infection when compared to experimental controls indicated that protection was conferred in mice during immunization. Our data shows that the parasite enzyme cysteine protease is a potential target which can further be exploited for precise drug targeting and vaccine development against malaria.
Keywords: Plasmodium berghei, Cysteine protease, Immunization, Protection, Parasite burden.
Malaria is a serious disease caused by parasites of the genus Plasmodium and it is transmitted by female anopheline mosquitoes.
Malaria poses a tremendous impact on human health, killing millions of people annually and it is a major impediment for social and economic development in malaria-endemic areas in sub-Saharan Africa . Hence, after tuberculosis and HIV, malaria constitutes the single greatest threat to human health of the recognized infectious diseases in terms of annual mortality and morbidity .
Due to the global resistance of malaria parasites to mainstay antimalarial drugs, there is need for new approaches to malaria treatments. Among these approaches is the targeting of enzymes with essential roles in the parasite's life cycle .
Proteases which appear to be required for a number of important functions in erythrocytic parasites, including haemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion are important in the pathophysiology of malaria . The best characterized proteolytic function is the hydrolysis of haemoglobin, which provides amino acids for the parasite's protein synthesis and other necessary functions .
In Plasmodium falciparum, the most virulent human malaria, haemoglobinases have been identified from the aspartic  cysteine  and metallo  protease classes. But the specific roles of these enzymes in the sequential hydrolysis of haemoglobin have not yet been delineated. Treatment with cysteine protease inhibitors blocks haemoglobin hydrolysis and parasite development in vitro  and in murine models , suggesting that Plasmodial cysteine proteases are appropriate chemotherapeutic targets .
The use of enzymes associated with protection against oxidative stress has been shown to be vaccine targets in leishmania . In the present study, we report the acquisition of protection in BALB/c mice by immunization with purified forms of cysteine protease against a lethal challenge with virulent strain of Plasmodium berghei.
Complete Freund Adjuvant and Incomplete Freund Adjuvant were obtained from the National Veterinary Research Institute (N.V.R.I) Vom, Jos-Nigeria. All other reagents were of analytical grade.
Mice and parasites
Age and sex matched BALB/c mice (20-25g) in-house breed at the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria- Nigeria were used in this study. They were maintained on food and water ad libitum at the animal house situated at the Department of Biochemistry, Ahmadu Bello University, Zaria-Nigeria. The guide for care and use of laboratory Animals, 1996 of the Institute of Laboratory Animal Research (ILAR) Commission on life Science, National Research Council was duly followed.
The chloroquine-resistant strain of the malaria parasite, Plasmodium berghei was obtained by Professor A.J Nok (MFR) from the Department of Parasitology, Kuvin Medical Centre, Hebrew University of Jerusalem, Ein-keren Israel. The strain was maintained during this study, in the laboratory by serial blood passage from mouse to mouse.
Infection and parasitaemia determination
Twelve (12) healthy albino mice were intraperitoneally infected by injection of 0.2 ml of infected blood diluted with sodium citrate solution to contain about 1 x106 parasitized red blood cells (pRBC) of virulent Plasmodium berghei parasites. Blood samples were collected by bleeding via the tail vein of P. berghei-infected mice and thin blood smears were made on microscope slides, fixed in methanol, and stained with 10% Giemsa solution (Merck, Tokyo, Japan). The percentage parasitemia was determined by counting the percentage pRBC for at least ten (10) different fields. At peak parasitaemia (70-75%), blood was collected in heparinized syringe by cardiac plexus puncture.
Phase separation of parasite proteins
Triton X-100 solubilization and phase separation procedures were conducted on freshly collected infected erythrocytes and lysed in sterilized distilled water . Briefly, 0.4 ml of 0.5 % TX-100 in 50 mM Tris-buffered Saline was dispensed into 10 ml of the parasite suspension and incubated at 4ºC for 90 minutes. The supernatant was collected after an initial centrifugation at 10,000 x g for 30 minutes at 4ºC and was layered onto a 6 % sucrose cushion containing 0.06 % TX-100 followed by incubation at 37ºC for 5 min. The aqueous and detergent phases were collected after centrifugation at 900 x g for 5 minutes at 37ºC and were respectively precipitated by acetone to recover the aqueous and integral membrane proteins respectively. The pellets from each preparation were suspended to 6 ml in 50 mM phosphate buffered saline, pH 7.2.
Total protein estimation
Protein was quantified using Bovine Serum Albumin (BSA) as standard .
Enzyme activity assays
The aqueous and detergent phase preparations of proteins were used for cysteine protease activity assays in the absence and presence of specific protease inhibitors.
Briefly, 50µl of the protein sample was added to 500µl of 100 mM sodium acetate buffer, pH 4.5, and 100µl of 3 % gelatin. The final volume of the reaction mixture was adjusted to 1ml with distilled water. Assays were carried out at 37ºC for 1hour and the reaction was stopped by the addition of 200µl of 20 % (v/v) trichloroacetic acid. After the removal of precipitated protein by centrifugation (10,000 x g for 5 min at room temperature), absorbance of the supernatant was measured at 366nm using a spectrophotometer .
One unit of proteolytic activity was defined as 1µmole of tyrosine hydrolyzed per hour under standard assay conditions.
Exactly five milliliters (5ml) of the reconstituted, crude parasite proteins from the detergent-treated phase was applied onto a DEAEcellulose column (1 cm X 12 cm) pre-equilibrated with 50 mM of Phosphate Buffer, pH 7.2 containing 1 mM dithiothreitol, after repeated washings with the operating buffer which removed any unbound material, the column was eluted with a step-gradient of NaCl (0.0-0.3M) in 50 mM phosphate Buffer. Twenty fractions were collected and assayed for CP and total proteins. Active fractions were pooled and applied onto a gel permeation chromatograhy column (Sephadex G-50, 1 cm X 12 cm) pre-equilibrated with 50 mM acetate buffer, pH 5. The protein was eluted isocratically with the operating buffer and thirty two (32) fractions, each of 2ml were collected. Fractions with high specific activities but with very close elution times were pooled resulting to two major peaks (CP-A and CP-B) which were analyzed by 12 % SDS-PAGE . CP-B which was exclusively sensitive to the cysteine protease specific inhibitor, iodoacetate was used for immunization studies.
Immunization and parasite challenge experiments
BALB/c mice weighing 20-25 g were used for immunization. Five groups of mice, five per group were immunized intraperitoneally with 50µg of the purified cysteine protease (CP-B) and 50µg each of crude parasite proteins from the hydrophobic and hydrophilic phases respectively with each antigen emulsified in CFA. A control group of mice were administered the adjuvant alone emulsified in Phosphate- Buffered Saline. Booster immunizations were given on days 14 and 21 with antigens emulsified in IFA. A week after the last booster immunization, mice were challenged with Plasmodium berghei infected RBC (106). A second control group of un-immunized mice, infected with the same inoculum size was included. The course of infection was monitored daily by microscopic Giemsa stained thin blood smears.
Pack Cell Volume (pcv) values were determined using the microhaematocrit method.
Microscopic detection of thin blood smears
Microscopic views of Giemsa-stained thin blood smears of infected mice showed that the malaria parasites were released from red blood cells when subjected to a lysis protocol (Figure 1).
Cysteine protease purification
The specific activity of Plasmodium berghei cysteine protease in the crude protein preparation and the purified cysteine protease from the crude parasite preparation were determined to be 0.895 and 4.770µmoles/min respectively. Analysis of the stable form of cysteine protease (cp-b) on 12 % polyacrylamide gel showed four (4) bands ranging from 20-47 kDa (Figure 2).
Protective efficacy of cysteine protease in mice
Mice immunized with adjuvant alone developed parasitemia by day 2 to 5 post -infection and reached a peak parasitemia of 67 % ± 2 on day 10 ± 2 and succumbed to infection (Figure 3), while mice immunized with purified cysteine protease formulated in adjuvant reached a peak parasitemia of 28.77% ± 0.87 on day 18 ± 4 (P=0.05). In comparison, mice immunized with crude parasite proteins from the hydrophilic and hydrophobic phases of the parasite had a week delay in the onset of parasitemia and mice were protected against challenge infection. This was shown by lower levels of peak parasitaemia of 44.22 % ± 1.11 on day 12 ± 4 (P=0.05) and 47.9% ± 1.39 on day 12 ± 4 (P=0.05) respectively (Figure 4). All of the naïve mice succumbed to the infection with pRBC intraperitoneally by day 10. The mice immunized with the crude parasite proteins from the hydrophobic and hydrophilic components of the parasite had a mean survival time of 24 ± 2 days and 21 ± 2 days respectively. While mice immunized with the purified cysteine protease had a mean survival time of 30 ± 2 days, mice immunized with adjuvant alone had a mean survival time of 8 ± 2 days.
Haematocrit pack cell volume assessment
Assessment of the Pack Cell Volume (PCV) of immunized mice indicates that mice immunized with the purified cysteine protease had a better PCV profile than mice immunized with the crude parasite proteins from both phases when compared with experimental controls which had a drastic reduction in PCV values (Figure 4).
Several bioactive molecules have been evaluated as vaccine candidates with promising results, such as cathepsin, proteinase [14,15], and the enzyme glutathione transferases [16,17].
Cysteine protease play important role in the pathogenesis of the malaria parasite but despite decades of research there remains controversy about their function in the malaria erythrocyte cycle [18,19].
In the present work, we show on protein electrophoregram, the possible existence of multiple forms of the enzyme cysteine protease which is consistent with previous reports on Plasmodium falciparum falcipains . The existence of multiple forms of parasite enzymes is thought to confer an evolutionary advantage for the parasite that may contend with the development of vaccines, new anti-malarials and drug pressure that may target the enzyme.
We also show that immunization with the purified cysteine protease prepared from the hydrophobic phase of the malaria parasite conferred the strongest protection against challenge with Plasmodium berghei in BALB/c mice than the crude parasite proteins preparation from the hydrophobic and hydrophilic phases of the parasite when compared to experimental controls.
Protection was shown by a drastic reduction in total parasite burden which is attributed to protection conferred in mice during immunization against the infection.
This observation is supported by a previous report on immunization with a specific antigen that elicits antibody response and interferes with the invasion process of red blood cells and parasite propagation .
It is our opinion that the observed reduction in parasite burden and the increase in mean survival time of immunized mice post infection could be attributed to antibodies or effector cells in the immune sera against the antigen which may be interfering with the processing and/ or maturation of surface proteins required for productive invasion and parasite establishment.
Also, we expected the crude antigens to have a greater number of epitopes but it failed to confer the strongest protection. A possible explanation could be the possible masking of protective epitopes by junk proteins in the crude phases of the parasite protein preparation which may be a limiting factor for the efficacy of the crude antigens.
Also, to substantiate our observation is a report on the protection of trypanosomosis by immunization with pure trypanosomal tubulin extracts .
We also observed that the degree of protection conferred in the cysteine protease immunized groups of mice varied even within the same group, indicating that the potency of cysteine protease as an immunogen can be affected by the genetics of the host.
PCV values of mice immunized with the purified cysteine protease were higher than that of the crude parasite proteins from both phases of the parasite. This further exemplifies that immunization with the purified cysteine protease was more effective in ameliorating anemic condition in infection when compared to experimental controls.
During the blood stage infection, a large number of schizonts rupture simultaneously and fever paroxysms sets in which results in the release of toxins like glycosylphosphatidylinositol structures which further stimulate the host to produce pyrogenic cytokines such as tumor necrosis factor. This has been implicated in ineffective erythropoiesis in murine malaria .
Anaemia results partly due to direct destruction of erythrocytes by parasites, erythrophagocytosis as well as dyserythropoiesis which is associated with Th-1 cytokines that initiates the diversion of iron necessary for the synthesis of hemoglobin to the storage compartments of macrophages in the reticuloendothelial system resulting in limited erythropoiesis, thus less iron is made available for heme synthesis .
Protection conferred in mouse models which we observed on immunization with the purified form of cysteine protease is likely associated with the acquisition of protective antibodies.
Although, we did not assessed antibody titres by ELISA but it can be said that high titres of antibodies were attained. This is evident in the cysteine protease immunized mice by the diminishing parasitemia load, increased mean survival time post-infection  and the prevention of drastic reductions in PCV values typical of the malaria infection.
Cysteine proteases from parasites have been shown to be immunogenic and are being exploited as serodiagnostic markers and vaccine targets [25,26]. Antibody-mediated protection has been reported with P. falciparum SERA/SERP H antigens in Aotus monkeys and mice [27,28], with Leishmania major cysteine protease in mice . The possibilities for an anti-cysteine protease vaccine against parasitic organisms have been reported also for Fasciola hepatica and Haemonchus contorius infections [30,31], as well as for Trypanosoma cruzi [32,33].
Furthermore, it has been demonstrated that blocking the expression of the cysteine protease genes in Plasmodium falciparum results in severe morphological abnormalities in the parasites as well as inhibition of parasite growth in vitro .
Even though a broad spectrum of cysteine protease inhibitors have been reported to block invasion by malaria parasites, this further suggests that the yet undiscovered cysteine proteases may function together to ensure successful invasion .
Our data certainly corroborates previous reports and forms a baseline for the exploration of anti-cysteine protease vaccines against the malaria parasites.
We thank the staff of Mary Hallaway Teaching Laboratory of the Department of Biochemistry, Ahmadu Bello University, Zaria-Nigeria for their technical assistance.