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Effect of Antibodies On The Expression of Plasmodium Falciparum Circumsporozoite Protein Gene
Effect of Antibodies On The Expression of Plasmodium Falciparum Circumsporozoite Protein Gene
2006, 3 7
control; b) 1mg/ml and 1μg/ml purified IgG Fab fragment incubated at RT for one hour. P. falciparum 3D7 48h culture
isolated from a pool of 750 malaria patients from Malawi, supernatant was centrifuged at 2000g to remove debris.
kindly provided by Prof. Hommel through Dr. Armada, Blocking buffer in the wells was replaced with 100μl of
Liverpool Scholl of Tropical Medicine, UK; c) 1 – 0.1 – 0.01 culture supernatant and incubated for 1h. Fresh culture
and 0.001 μg/ml anti-CSP monoclonal antibody (2A10- medium was used as negative controls. For each plate a
HA2) kindly provided by Dr. Wirtz, CDC, Atlanta, Georgia titration (100 – 0.75pg) of recombinant positive control (Pf-
USA. Parasites were cultured for 24 or 48h in a CO2 rich PC, lot R32, CDC, Atlanta) was added. The wells were then
environment. Blood smears were performed at 24 and 48 h. washed twice with PBS Tween20. Peroxidase conjugate
After 48h culture media and cells were collected for ELISA antibody diluted in blocking buffer (0.05μg) was added to
and RNA extraction respectively. each well. After 2h of incubation, plates were washed 3
RNA preparation and cDNA synthesis times with PBS Tween20, and peroxidase substrate added.
The absorbance was read at 414 nm. The standard curve
Extraction of total RNA was performed with Trizol was used to quantify the amount of CSP present in the
(Life Technologies) according to the manufacturer’s samples. ELISA mAbs and positive control were acquired
protocol. One microgram of total RNA from each sample from Dr. Wirtz, CDC, Atlanta, GA USA.
was treated with 1U of DNase I (Life Technologies).
Complementary DNA (cDNA) was synthesised in a 20 µl Statistical analyses
reaction with 1 µg treated RNA, 0.5μg oligo(dT)15, 50mM Data analysis was performed using SPSS statistical
Tris-HCl pH 8.3, 75mM KCl, 1,5mM MgCl2, 10mM BSA, program. The Wilcoxon signed-rank test, for 2 matched
0.5mM dNTP’s, and 10U MMLV-RT (Life Technologies). samples was used to evaluate differences between groups.
RNA was retrotranscribed for 1 hour at 37ºC, denatured 5 The significant level was taken as p<0.05.
min at 95ºC and quenched on ice. To certify the absence of 3. Results
genomic DNA (gDNA), duplicates of samples were
incubated with a similar cDNA reaction mixture devoid of At starting of experiments parasitaemia was set to
RT enzyme. No RNA controls were included for each approximately 2% PE. Blood smears were performed to
reaction mixture used. confirm parasitaemia that varied from 1.9 to 2.1
(percentage of ring stages/ early trophozoite varied 87 to
Real-Time Reverse Transcriptase-PCR Analysis 97%). After 48h of culture all controls showed an increase
Gene-specific primers for P. falciparum ldh [9] and csp in parasite number with total parasitaemia varying from
[10] genes were synthesised by Life Technologies and used 2.9 to 4.1. There were no statistical significant differences
in a reaction mixture using SYBR® Green PCR Core between control and treated parasites. However, we
reagents kit (PE Applied Biosystems). PCR reactions were observed a decrease in the mean number of parasites when
performed in a volume of 20µl containing the following treated with 1μg mAb/ml. Anti-CSP-mAb had
primer concentrations: ldh (0.3mM -fwd, 0.05mM-rev), csp contradictory effect depending on concentration and
(0.1mM -fwd, 0.3mM-rev). Each reaction contained 1µl of although mAb at lower concentration seems to have a
cDNA template. Amplification and detection of specific stimulatory effect, in this group the variability between
products was performed with the GeneAmp ® 5700 system experiments was high. The opposite was observed in
(PE Applied Biosystems) using the following cycle profile: parasites treated with IgG (Fig. 1).
1 cycle at 48°C for 30 min, 1 cycle at 95°C for 10 min, 40
cycles at 95°C for 15 s, and 60°C for 1 min. Figure 1. P. falciparum 3D7 in vitro growth. Parasites were
Quantification relies on the comparison of the critical submitted for 48h to different antibody treatments: 1μg/ml and
threshold cycle (Ct) of an unknown sample against a 0.001μg/ml anti-CSP monoclonal antibody (mAb); 1mg/ml and
standard curve of known quantities. Ct is the amplification 1μg/ml purified IgG Fab fragment isolated from a pool of 750
cycle at which the fluorescence becomes detectable and is malaria patients from Malawi. Cultures were set for 2%
inversely proportional to the logarithm of the initial parasitaemia (87-97 % ring stage parasites) and 5% heamatocrit
and were cultured for 48 hours. Percentage of parasite
amount of template DNA. For each reaction a standard
increase/decrease was calculated by dividing parasitaemia of
curve was plotted with Ct values obtained from treated cultures over control cultures (control = 100%).
amplification of known quantities of gDNA (100, 10, 1, 0.1 Parasitaemia of 3 replicates per experiment was counted. Data
and 0.01ng) isolated from P. falciparum 3D7 clone. represents mean increase of ring stage parasites ( - ) and
Concentration of DNA was determined by schizonts (+) of 5 independent experiments. Boxes represent
spectrophotometric analysis of optical density at 260nm standard error of the mean [open – ring stages; solid – schizonts]
(GeneQuant, Pharmacia) and standard DNA solutions and whiskers represent standard deviation of the mean.
were prepared. A standard curve was used to transform Ct
values to the relative number of DNA molecules.
Triplicates of each sample and standard curve were
performed in all assays. The quantity of cDNA for csp was
normalised to the quantity of the house keeping gene ldh
cDNA in each sample. Melting curves were used to
determine the specificity of PCR products.
ELISA for detection of circumsporozoite protein (CSP)
Microtitration plates were coated with PBS containing
0.2μg/well capture antibody (Pf-CAP, lot. TF097, CDC,
Atlanta) and incubated overnight at room temperature.
Coating solution was replaced with blocking buffer (0.5%
Casein, 0.1N NaOH, PBS pH7.4, 20mg/l phenol red) and
Int. J. Med. Sci. 2006, 3 9
P. berghei blood stage development, as disruption of csp 3. Vaughan JA, Do Rosario V, Leland P, et al. Plasmodium falciparum;
gene had no effect on blood stage infection [15], our data ingested anti-sporozoite antibodies affect sporogony in Anopheles
suggests that CSP might play a role during the erythrocytic stephensi mosquitoes. Exp Parasitol 1988; 66: 171-182.
development of the parasite. 4. Hollingdale MR, do Rosario V. Malaria transmission-enhancing
activity in mosquitoes by mammalian host anti-sporozoite antibodies.
Several functions have been described for CSP,
Exp Parasitol 1989; 68: 365-368.
sporozoite gliding motility [16] recognition and binding to
5. Peiris JS, Premawansa S, Ranawaka MB et al. Monoclonal and
the salivary glands [17] and more recently Thathy et al. [18] polyclonal antibodies both block and enhance transmission of human
have demonstrated that CSP is essential for sporozoite P vivax malaria. Am J Trop Med Hyg 1988; 39: 26-32.
morphogenesis in the oocyst. The effect observed at the 6. Nudelman S, Renia L, Charoenvit Y, et al. Dual action of anti-
present work indicates that availability of CSP during sporozoite antibodies in vitro. J Immunol 1989; 143: 996-1000.
erythrocytic stages can interfere with parasite 7. Shi YP, Udhayakumar V, Oloo AJ, et al. Differential effect and
multiplication and invasion, suggesting that CSP is likely interaction of monocytes, hyperimmune sera, and immunoglobulin G
to be involved indirectly on erythrocyte invasion. The on the growth of asexual stage Plasmodium falciparum parasites. Am J
immune localisation of the CS-like protein described by Trop Med Hyg 1999; 60: 135-141.
Cochrane et al. [12] only found at the micronemes of 8. Trager W, Jensen JB. Human malaria parasites in continuous culture.
Science 1976; 193: 673-675.
merozoites of mature invasive blood stages is also
9. Moormann AM, Hossler PA, Meshnick SR. Deferoxamine effects on
indicative that CSP might play a role in the early stages of Plasmodium falciparum gene expression. Mol Biochem Parasitol 1999;
parasite interaction with the host red blood cell. 98: 279-283
Antibody dependent enhancement of viral 10. Alloueche A, Silveira H, Conway DJ, et al. High-throughput sequence
replications has been described in several viruses typing of T-cell epitope polymorphisms in Plasmodium falciparum
(reviewed by Sullivan [19]). This phenomenon has been circumsporozoite protein. Mol Biochem Parasitol 2000; 106: 273-282.
associated, with the ability of viruses to interfere with the 11. Cohen S, McGregor IA, Carrington SC. Gamma-globulin and acquired
activity of antiviral pathways, cytomegalovirus inhibited immunity to human malaria. Nature 1961; 192: 733-777.
class II major histocompatibility complex expression in 12. Cochrane AH, Uni S, Maracic M, et al. A circumsporozoite-like protein
endothelial cells and fibroblasts [20] and Ross River virus is present in micronemes of mature blood stages of malaria parasites.
Exp Parasitol 1989; 69: 351-356.
interfered with transcription of key antiviral genes through
13. Levitt A, Dimayuga FO, Ruvolo VR. Analysis of malarial transcripts
targeting the transcription factors IRF-1 and NF-κB [21]. As using cDNA-directed polymerase chain reaction. J Parasitology 1993;
virus relies on host cell machinery for multiplication, anti- 79: 653-662.
viral antibodies exert their effect on host cell. In malaria, 14. Ruvolo V, Altszuler R, Levitt A. The transcript encoding the
the presence of CSP in the cytoplasm of HepG2 cells and circumsporozoite antigen of Plasmodium berghei utilizes heterogeneous
macrophages has been described to inhibit protein polyadenylation sites. Mol Biochem Parasitol 1993; 57: 137-150.
synthesis in the target cells, through interaction with 15. Menard R, Sultan AA, Cortes C, et al. Circumsporozoite protein is
ribosomes [22]. Based on their observations the authors required for development of malaria sporozoites in mosquitoes.
suggested that malaria sporozoites are able to control the Nature 1997; 385: 336-340.
protein synthesis machinery in cells of the vertebrate host, 16. Stewart MJ, Vanderberg JP. Malaria sporozoites release
which could explain the differences in the levels of circumsporozoite protein from their apical end and translocate it
along their surface. J Protozool 1991; 38: 411-421.
expression observed in our study. 17. Sidjanski SP, Vanderberg JP, Sinnis P. Anopheles stephensi salivary
The effect of antibodies directed to an antigen glands bear receptors for region I of the circumsporozoite protein of
predominantly expressed in a different life cycle stage, Plasmodium falciparum. Mol Biochem Parasitol 1997; 90: 33-41.
could contribute to parasite escape from destruction by 18. Thathy V, Fujioka H, Gantt S, et al. Levels of circumsporozoite protein
influencing the efficacy of mechanisms such as host cell in the Plasmodium oocyst determine sporozoite morphology. EMBO J
invasion. Parasite manipulation of their expression 2000; 21: 1586-1596.
machinery by host immune system molecules might be a 19. Sullivan NJ. Antibody-mediated enhancement of viral disease. Curr
parasite mechanism that evolved to avoid potential lethal Top Microbiol Immunol 2001; 260: 145-169.
effects of neutralising Ab. 20. Miller DM, Rahill BM, Boss JM, et al. Human cytomegalovirus inhibits
major histocompatibility complex class II expression by disruption of
Acknowledgements the Jak/Stat pathway. J Exp Med 1998; 187: 675-683
We are grateful to Claudia Melo for performing the 21. Lidbury BA, Mahalingam S. Specific ablation of antiviral gene
CSP – ELISAs. We would like to thank Prof. Hommel and expression in macrophages by antibody-dependent enhancement of
Ross River virus infection. J Virol 2000; 74: 8376-8381.
Dr. Armada for providing purified IgG Fab fragment. CSP
22. Frevert U, Galinski MR, Hugel FU et al. Malaria circumsporozoite
detection ELISA was provided by Dr. Wirtz, CDC, Atlanta, protein inhibits protein synthesis in mammalian cells. EMBO Journal
GA, USA. We would also like to thank Encarnação Horta 1998; 17: 3816-3826.
for technical assistance.
This work was supported by research funds from the
project POCTI/35815/MGI/2000.
Conflict of interest
The authors have declared that no conflict of interest
exists.
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