vant (FCA) and challenged with 1 x 10 live P. berghei showed that SIII imparted complete protection against infection. SIII with FCA or saponin adjuvants showed ...
A 24,000 g sediment of Plasmodium berghei induces IL-1 response in mice and exhibits protection against malaria infection
Javed Agrewala U P M A
l
1
and H. S. B A N Y A L
2
IMTECH, Chandigarh, 160014, India
Department ofZoobgy, Panjab University, Chandigarh, 160014, India (Received February 25,1998) Abstract'.Plasmodium berghei was subjected to differential centrifugádon. Mice immunized with sediments (SI and SHI) emulsified in Freund's complete adju vant (FCA) and challenged with 1 x 10 live P. berghei showed that SIII imparted complete protection against infection. SIII with FCA or saponin adjuvants showed the latter to be a better adjuvant as postchallenge parasitaemia in mice remained below 2%. Immune sera obtained from the Slll-saponin group inhibited 89% merozoite invasion in vitro. High antibody level was detected by indirect im munofluorescence assay (IFA). Sera collected from S-III immunized mice showed enhanced level of IL-I. Biochemical analysis of sediments revealed SIII to be enriched in acid proteinase and acid phosphatase. The role of parasite com ponent sedimented at 24,000 g in malaria protection is discussed. Key words: Plasmodium berghei, malaria, in vitro, IL-I, saponin
INTRODUCTION Clinical-manifestations of malaria are mainly due to blood stages of Plasmodium. Merozoite invades the host's erythrocyte and propagates intracellularly by utilizing host cell haemoglobin (Pasvol and Wilson 1982). Invagination of red cell membrane is mediated by contents of rhoptries emptied during the process of invasion (Bannister et al. 1986). A high molecular weight protein present in the rhoptry of P. yoelii exhibited protective immunity (Holder and Freeman 1981). The rhoptry content involved in invasion process may be analogous to the proteases of parasite since a 68 kDa neutral endopeptidase of P. berghei has been associated with the apical complex of the merozoite (Bernard and Schrevel 1987). Studies with protease inhibitors have also suggested a definite role of malarial protea ses in the invasion process (Banyai et al. 1981; Hadley et al. 1983). Proteases degrade host cell haemoglobin and are present in lysosomal organelles in Plasmodium (Vander Jagt et al. 1992). Sephadex purified malarial proteases partially protect mice against P. berghei (Makkar et al. 1995).
Various antigens have been isolated from different constituents of Plasmodium and these antigens have exhibited a protective role against malaria (Newbold 1984). The parasite constituents can be separated by differential centrifugation (Banyai et al. 1979), however, the role of the various cellular organelles in the protection against malaria has not been elucidated. In this study a fraction of P. berghei separated by centrifugation induced protective immune response in mice.
MATERIALS AND METHODS Parasite: P. berghei (NK-65) asexual erythrocytic stages were maintained in white Swiss mice (Balb/C), 20-25 g of either sex. When the parasitaemia was 50%-60%, blood fromjR berghei-infectcd mice was collected in citrated saline and passed through CF-11 cellulose (Whatmann). Erythrocytes were washed with phosphate buffered saline (0.01 M , PBS, p H 7.2) and cell-free parasites were isolated by saponin lysis (0.2% w/v) followed by centrifugation at 10,000 g for 20 min. The cells were washed twice with PBS, p H 7.2. Cell-free parasites were suspended in an appropriate volume of 0.25 M sucrose solution and homogenised in a Potter Elvehjam precooled homogenizer (Remi, Bombay, India) at 2000 rpm with fifty strokes of pestle. Homogenate ( H O M ) was centrifuged at 600 g for 15 min at 4 °C. The sediment designated SI was obtained and the supernate was further centrifuged at 10,000 g for 25 min. The sediment (SII) was isolated and the supernate again centrifuged at 24,000 g for 35 min. The sediment (SIII) was separated from the supernate fraction (SIV). The method of Banyai et al. (1979) was followed for differential centrifugation. A l l centrifugations were carried out at 4 °C in a refrigerated centrifuge. A part of the sediment was suspended in normal saline and used as enzyme for biochemical analysis. Rest of the sediments were suspended in PBS (0.01M, p H 7.2) and used for immunizing mice. Protein was estimated by Folin-phenol reagent (Lowry et al. 1951). Immunization of mice : I n a typical experiment ten mice were immunized in each group with H O M , SI, and SIII. Each mouse of a group received 100 fig protein of the respective fraction emulsified in Freund's complete adjuvant (FCA) on day 0 subcutaneously (s.c.) followed by the same amount of protein in PBS, p H 7.2 intraperitoneally (i.p.) on day 14 and day 28. Mice of the control group were injected with PBS, p H 7.2 and F C A on day 0 followed by only PBS, p H 7.2 on day 14 and day 28. On day 35, seven mice from each group were injected i.p. with 1 x 10 P. berghei-inîecteâ red cells and the remaining mice of each group were bled to collect immune sera (precha llenge). Postchallenge parasitaemia from each mouse was recorded daily by preparing Giemsa-stained blood smears. Percent infection was calculated as: number of infected red cells JQQ total number of red cells 6
x
Mice which survived the challenge were bled to collect postchallenge immune sera after their smears were clear from infection. In a different experiment another four groups of 10 mice each were immunized in the following way. Each mouse of a group (SHI-saponin), was injected i.p. with 100 pg of SIII protein in PBS, p H 7.2 containing 30 pg saponin each on days 0,14 and 28.
The other group of mice (SIII-FCA) was immunized with SIII protein emulsified in FCA as explained above. The third and fourth groups were used as controls for saponin and F C A respectively. Each mouse of these last two groups received an equal amount of saponin or F C A as expérimentais without any parasite protein on days 0,14 and 28. On day 35 mice (experimental as well as control) were inoculated with 1 x 10 JR berghei parasitized red cells and the postchallenge course of parasitaemia was recorded as explained earlier. 6
6
Four immune mice of the SHI-saponin group were rechallenged with l x 10 P. berghei infected erythrocytes after the first challenge infection was cleared from their blood. Antibody titration by indirect immunofluorescence assay (IFA): Immune serum (pre- and postchallenge) samples were diluted in 2-fold steps starting at 1:16, in PBS p H 7.2. These sera were titrated on acetone-fixed P. berghei antigen slides according to Collins and Skinner (1972). The second antibody employed was rabbit anti-mouse IgG conjugated to F I T C (Sigma, U S A ) . Slides were counterstained in 0.5% (w/v) Evans blue in PBS, p H 7.2. The end point was defined as the highest serum dilution showing no fluorescence. Merozoite invasion inhibition assay: The short-term in vitro culture of P. berghei was carried out in 24-well culture plates. Each well contained 1.0 m l of complete medium (RPMI-1640) supplemented with gentamycin (50 pg/ml), penicillin (100 IU/ml), streptomycin (100 pg/ml), sodium bicarbonate (5%, w/v) and N-2-hydroxyethyl piperazine N - 2 ethane sulphonic acid (HEPES) (0.6% w/v) and 10% (v/v) heat-inactivated (56 °C, 30 min) fetal calf serum. Normal mouse serum (5%, control) or immune mouse serum (5%, experimental) and normal and schizont/trophozoite infected ( l % - 2 % parasitaemia) erythrocytes (2% haematocrit) were added to the medium. Cultures were placed in a candle jar and incubated at 37 °C. After 19 h of incubation smears were prepared using the well contents, fixed in methanol, stained in Giemsa's stain, and the percent inhibition of merozoite invasion was calculated as follows: . number of rings in experimental culture _ % inhibition = 100 • f-. :—: x 100 number of rings in control culture Detection of interleukin (IL-1): Sandwich enzyme immunoassay (Ferma et al. 1988) was performed to measure the level of I L - 1 in pre- and postchallenge immune sera raised against sediment (SIII). For the assay, 96-welI microtitre plates were coated with 50 p i of rabbit anti-mouse I L - 1 (5 pg/ml) and incubated overnight at 4 °C. After washing with PBS, 0.01 M , p H 7.2 - Tween 20, unbound sites were saturated with 100 p i of 2% (w/v) bovine serum albumin (BSA) in PBS, p H 7.2 for 1 h at 37 °C. Thereafter, plates were washed three times with PBS-Tween 20. Fifty p i aliquots standards (recombinant IL-1) and different dilutions (multiples of log 5) of immune sera (diluted in 0.5% BSA in PBS) were dispensed into duplicate wells of microtitre plates and incubated overnight at 4 °C. After aspirating the unbound material and washing with PBS-Tween 20, 50 p i of goat anti-mouse I L - 1 was added to the wells and plates were incubated at 37 °C for 2 h. This was followed by washing of plates three times with PBS-Tween 20. Thereafter, 50 p i of anti-goat Ig-HRP (horse radish peroxidase) labelled antibody (1 : 2500) was added and incubated at 37 °C for 2 h. After aspirating unbound conjugate, plates were washed five times. Fifty p i of orthophenylenediamine (OPD) was added in dark to each well and the plates were ;
n
n
t
n
n
incubated at 37 °C for 20 min. The reaction was stopped with 50 p i of 7% (v/v) sulphuric acid and plates were read at 492 nm in an ELISA reader (Eurogenetics, model M P R A4, Torino, Italy). The data were calculated from the mean of duplicate determinations as units/ml as computed by comparison with the standard curve using recombinant IL-1 (Genzyme, U.S.A). Acid protease assay: Acid protease assay was performed using 2.5 m l reaction mixture containing 0.1 M acetate buffer, p H 4.6,0.1% (w/v) haemoglobin (denatured) and 0.1 ml enzyme (Banyai et al. 1982). Tyrosine released in protein-free supernate was measured by Fohn-phenol method (Lowry et al. 1951). One unit of enzyme was the amount of enzyme that liberated one mole of tyrosine in 60 min at 37 °C. Acid phosphatase assay:The 2.5 ml assay mixture of acid phosphatase contained 0.1 ml of enzyme, 18 m M sodium B-glycerophosphate, 20 m M sodium barbitone and 0.1 M acetate buffer, p H 5.0 (Banyai et al. 1980). Phosphorus released after incubation at 37 °C for 60 min was measured by the method of Fiske and Subba Row (1925). Alkaline phosphatase assay: The assay mixture contained similar amount of substrate and sodium barbitone as in the acid phosphatase assay but without acetate buffer and the p H of the substrate solution was raised to 8.9 with 0.1 N N a O H . Phosphorus released was measured as above and one unit of enzyme (acid and alkaline phosphatase) activity was the amount of enzyme which liberated one mole of phos phorus in 60 min at 37 °C. The protein content of samples was estimated by the Folin-phenol method (Lowry et al. 1951). The specific activity of an enzyme was expressed as units of enxyme activity per milligram protein. RESULTS Figure 1 represents the course of parasitaemia in mice immunized with homogenate (Horn) and sediments SI, SIII along with controls in a typical experiment. A l l the mice of control group and the SI group died because of infection. Mice in the H O M and SIII groups survived but the former group had higher parasitaemia (10-20%) followed by clearing of infection. However, the latter group had very little infection (1-8%) which was subsequently cleared off. Mice were also immunized using SII and SIV sediments. A l l SIV mice died upon challenge, whereas some of the SII mice survived the challenge after having high parasitaemia (30%) (data not shown). A comparable study using SIII with F C A (SIII - FCA) and saponin (Slll-saponin) adjuvants was also carried out (Fig. 2). A l l the mice immunized with SIII using both the adjuvants were protected upon challenge. The maximum postchallenge parasitae mia ranged between 5%-7.1% and 0.2%-1.7% in SIII-FCA and Slll-saponin groups respectively. The results depicted in Figs 1 and 2 are of a typical experiment; however, a similar pattern of protection and course of parasitaemia in postchallenge mice was observed in repeated experiments. The level of antimalarial antibodies in immunized mice before (prechallenge) and after challenge (postchallenge) was measured by IFA (Fig. 3). No false reaction was observed because of counterstaining of slides with Evans blue. Prechallenge immune sera of the H O M - F C A and SIII-FCA groups had a high level of antibodies which was further boosted after challenge. However, with SI a low antibody titre was recorded.
100-,
OAYS Fig. 1. Course of parasitaemia in mice immunized with total parasite and various sediments of Plasmodium berghei and subsequently challenged with 1 x 10 P. berghei infected red cells. Bar represents mean standard deviation (STD). (•) Placebo controls, (O ) HOM-total parasite homogenate, ( x ) S I sediment of P. berghei after centrifugation of Horn at 600 g for 15 min. and (A ) Sill sediment of 24,000 g. 6
Fig 2. Course of parasitaemia in mice immunized with SIII from P. berghei using saponin and Freund's complete (FCA) adjuvants and challenged with 1 x 10 P. berghei infected red cells. Bar represents mean STD. (•) normal control, (o) placebo control, ( x ) SIII - FCA, (A) Slll-saponin. 6
SOOO-i
Conrol
SERA Fig. 3. Antimalarial antibody titre in im munized mice determined by indirect im munofluorescence assay • - prechallenge serum, M - postchallenge serum. A - ref erence malaria positive, B - HOM, C SI and D - SIII.
0 K
Horn
SI
SB
After 19 h
Fig. 4. In vitro inhibition of invasion of erythrocytes by P. berghei in the presence of prechallenge sera from immunized mice. Bar represents mean STD. Results are mean of four experiments. • - ring, M • trophozoi te, H - schizont.
The antiparasitic effect of antibodies was also evaluated in vitro using a short-term culture of P. berghei. In vitro invasion of normal erythrocytes by P. berghei was inhibited in the presence of immune sera (Fig. 4). Prechallenge serum from mice immunized with the SIII fraction inhibited about 89% of the invasion, whereas, total parasite homogenate ( H O M ) inhibited 75% invasion. Agglutination of some trophozoites and schizonts was observed in culture smears and a few merozoites were also found attached to erythrocytes. The prechallenge sera of mice immunized with SI did not show any significant inhibition of invasion. Concentration of interleukin (IL-1) in prechallenge immune sera of the SIII-FCA fraction was considerably high (305.0 pg/ml) as compared to controls (pg/ml). However, concentration of I L - 1 in postchallenge immune serum came down to normal ( pg/ml). The differential centrifugation of total parasite homogenate resulted in various sediments. These sediments were analysed for the activity of different marker enzymes (Table 1). SIII (24,000 g pellet) contained maximum activity of acid phosphatase and acid protease while alkaline phosphatase activity was maximum in SI. The data given are of three independent preparations and the maximal activity of the marker enzymes was observed in their respective fractions. Table 1 Specific activity of marker enzymes in total parasite homogenate of P. berghei and its different sediments (results are the mean of three experiments, each run in duplicate along with appropriate controls)
Enzyme (units/mg protein) Fraction
Acid protease
Acid phosphatase
Alkaline phosphatase
Horn
1.296±0.009
11.101±0.050
2.360±0.003
SI
0.938±0.003
2.172±0.009
18.170±0.910
SII
0.894±0.010
0.992+.0.002
1.730+0.001
SIII
5.573±0.091
13.840±0.090
4.379±0.003
SIV
1.100+0.040
1.40910.003
2.05U0.090
DISCUSSION SIII, the sediment obtained after centrifugation at 24,000 g of P. berghei, induced immune response in mice against the parasite, and all the immunized animals were protected upon challenge. The immune response appears to be both humoral and cellular. Humoral immunity is evident from the observations of high antibody titre determined by IFA (Fig. 3) and significant blocking of host cell invasion by merozoites in vitro resulting in a lower number of new rings formed and reduced propagation of parasite in the presence of immune serum (Fig. 4). As compared to the normal mice the high concentration of I L - 1 in the sera of immunized animals and subsequent protection of these animals upon challenge shows that SIII elicited also a cellular response.
The SIII fraction contained high activity of acid protease (specific activity 5.573± 0.091 ) and acid phosphatase (specific activity 13.840+0.090). In addition to host cell haemoglobin degradation the proteases from Plasmodium (Goldberg et al. 1990; Bailly et al. 1991) are also involved in the invasion of erythrocytes by merozoites (Banyai et al. 1981 and Hadley et al. 1983). I t appears that in vivo protection exhibited by SIII may be mediated by the humoral antibodies induced by proteases present in the SIII as its immune serum exhibited an invasion-blocking effect in vitro (about 89% inhibition). In our laboratory, we have observed that mice immunized with Sephadex G-200 purified acid protease from P. berghei were partially protected upon challenge and antiprotease antibodies inhibited in vitro invasion of mouse erythrocytes by P. berghei (Makkar et al. 1995). Antiprotease antibodies have also been indicated to inhibit propagation oîP berghei (Bernard and Schrevel 1987). Our studies with SIII have shown that the antigenic component isolated from this by immunoadsorption contained 66 kDa and 79 kDa proteins and exhibited proteolytic activity, and mice immunized with this component were protected upon challenge (Upma and Banyai, manuscript in preparation). Further, the mice immunized with SIII containing 30 pg of saponin were protec ted upon challenge (Fig. 2). The maximum parasitaemia in these mice was less as compared to SIII-FCA immunized mice, showing that saponin appeared to be a more potent adjuvant of the two (Freeman and Holder 1983). The cellular response induced by SIII is indicated by the presence of a higher level of I L - 1 in the prechallenge sera. Cytokines play an important role in malaria immunity. Exogenous administration of 80 ng of I L - 1 protected the mice against cerebral malaria and also delayed the onset of parasitaemia in P. berghei infection (Curfs et al. 1990). I n the present study, an enhanced level of I L - 1 (305 pg/ml) was observed in the sera of prechallenged mice as compared to the controls. These mice were protected upon challenge with live parasite and showed very low parasitaemia. This indicates that the SIII induced the production of I L - 1 in immunized animals and the protection achieved might have also been mediated by this cytokine as the level of I L - 1 fell considerably in postchallenge sera. The present study thus indicates that SIII of P. berghei, having high activity of lysosomal marker enzymes, induces a protective immune response in mice. The host red cell contami nation, if any, becomes irrelevant as the antibodies to red cells in mice would have resulted in agglutination of normal red cells in vitro invasion assay; however, no agglutination of the normal red cells was observed and in vivo also the antibodies to host red cell antigens may not have protected the mice against malaria infection as immunity to malaria is species as well as stage specific (Jeffery 1966; Newbold 1984). A component of SIII isolated by immunoadsorption, i.e. 66 kDa and 79 kDa protein, is protective against P. berghei infection (Upma and Banyai, manuscript in preparation), while the other constituents of the parasite, - in this case SII - did not induce antibodies in mice or protected the animals. Thus, SIII represents a promising target as protective antigenic material in malaria infection. Further elucidations are required as to whether this sediment containing high acid phosphatase activity (a marker of lysosomes) is enriched with lysosomal organelles/food vacuoles or certain other components of P. berghei which act as protective antigens. ACKNOWLEDGEMENTS Dr. Upma is thankful to U . G. C , New Delhi, for the financial assistance.
Upma, J. A. és Banyai, H. S.: A Plasmodium berghei-ből nyert 24,000 g-os üledék I L - 1 választ indukál egérben és véd a maláriás fertőzéssel szemben A szerzők egereket immunizáltak a Plasmodium berghei szakaszos centrifugálásából kapott üledékkel (SI és SIII). Az SIII üledék teljes védelmet indukált a fertőzéssel szemben. Az SIII saponinos emulziója bizonyult a legjobbnak, a beoltott egerekből származó szérum in vitro 89 %-ban gátolta a merozoiták invázióját és a szérumban emelkedett az I L - 1 szint. A szerzők megvitatják a 24 000 g parazita komponens szerepét a malária elleni védelemben.
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