Inhibition of Sporozoite Invasion

The Double-Staining Assay
  • Laurent Rénia
  • Ana Margarida Vigário
  • Elodie Belnoue
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 72)


During the life cycle of the malaria parasites in mammals, sporozoites invade hepatocytes, and give rise to thousands of merozoites that are able to infect red blood cells. Invasion of the liver cells is thus a critical step by which infection becomes established in the host, and against which an antiparasite attack might be effective. The development of hepatic cell culture systems (1-7) has made it possible to study several aspects of the liver stage infection, including penetration of the hepatocyte by the sporozoite (8-13).


HepG2 Cell Human Hepatocyte Hepatocyte Culture Infected Mosquito Hepatocyte Membrane 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Hollingdale, M. R., Leef, J. L., McCullough, M., and Beaudoin, R. L. (1981) In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei. Science 213, 1021–1022.CrossRefPubMedGoogle Scholar
  2. 2.
    Hollingdale, M. R., Leland, P., and Schwartz, A. L. (1983) In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei in a hepatoma cell line. Am. J. Trop. Med. Hyg. 32, 682–684.PubMedGoogle Scholar
  3. 3.
    Mazier, D., Landau, I., Miltgen, F., Druilhe, P., Lambiotte, M., Baccam, D., and Gentilini, M. (1982) Infestation in vitro d’hépatocytes de Thamnomys adulte par des sporozoites de Plasmodium yoelii: schizogonie et libération de mérozoites infestants. C. R. Acad. Sci. Paris. 294, 963–965.Google Scholar
  4. 4.
    Mazier, D., Landau, I., Druilhe, P., Miltgen, F., Guguen-Guillouzo, C., Baccam, D., et al. (1984) Cultivation of the liver forms of Plasmodium vivax in human hepatocytes. Nature 307, 367–369.CrossRefPubMedGoogle Scholar
  5. 5.
    Mazier, D., Beaudoin, R. L., Mellouk, S., Druilhe, P., Texier, B., Trosper, J., et al. (1985) Complete development of hepatic stages of Plasmodium falciparum in vitro. Science 227, 440–442.CrossRefPubMedGoogle Scholar
  6. 6.
    Millet, P., Collins, W. E., Fisk, T. L., and Ngyuen-Dinh, P. (1988) In vitro cultivation of exoerythrocytic stages of the human malaria parasite Plasmodium malariae. Am. J. Trop. Med. Hyg. 38, 470–473.Google Scholar
  7. 7.
    Millet, P., Fisk, T. L., Collins, W. E., Broderson, J. R., and Ngyuen-Dinh, P. (1988) Cultivation of exoerythrocytic stages of Plasmodium cynomolgi, P. knowlesi, P. coatneyi, and P. inui in Macaca mulatta hepatocytes. Am. J. Trop. Med. Hyg. 39, 529–534.PubMedGoogle Scholar
  8. 8.
    Cerami, C., Frevert, U., Sinnis, P., Takacs, B., Clavijo, P., Santos, M. J., and Nussenzweig, V. (1992) The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites. Cell 70, 1021–1033.CrossRefPubMedGoogle Scholar
  9. 9.
    Frevert, U., Sinnis, P., Cerami, C., Shreffler, W., Takacs, B., and Nussenzweig, V. (1993) Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes. J. Exp. Med. 177, 1287–1298.CrossRefPubMedGoogle Scholar
  10. 10.
    Sinnis, P., Clavijo, P., Fenyo, D., Chait, B. T., Cerami, C., and Nussenzweig, V. (1994) Structural and functional properties of region II-plus of the malaria circumsporozoite protein. J. Exp. Med. 180, 297–306.CrossRefPubMedGoogle Scholar
  11. 11.
    Müller, H.-M., Reckmann, I., Hollingdale, M. R., Bujard, H., Robson, K. J. H., and Crisanti, A.(1993) Thrombospondin related anonymous protein (TRAP) of Plasmodiumfalciparum binds specifically to sulfated glycoconjugates and to HepG2 Hepatoma cells suggesting a role for this molecule in sporozoite invasion of hepatocytes. EMBO J. 12, 2881–2889.PubMedGoogle Scholar
  12. 12.
    Pancake, S. J., Holt, G. D., Mellouk, S., and Hoffman, S. L. (1992) Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates. J. Cell Biol. 117, 1351–1357.CrossRefPubMedGoogle Scholar
  13. 13.
    Goma, J., Rénia, L., Miltgen, F., and Mazier, D. (1996) Iron overload increases hepatic development of Plasmodium yoelii in mice. Parasitology 112, 165–168.CrossRefPubMedGoogle Scholar
  14. 14.
    Hollingdale, M. R., Zavala, F., Nussenzweig, R. S., and Nussenzweig, V. (1982) Antibodies to the protective antigen of Plasmodium berghei sporozoites prevent entry into cultured cells. J. Immunol. 128, 1929,1930.Google Scholar
  15. 15.
    Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L., and Nussenzweig, R. S. (1984) Inhibition of entry of Plasmodium falciparum and P. vivax sporozoites into cultured cells: an in vitro assay of protective antibodies. J. Immunol. 132, 909–913.PubMedGoogle Scholar
  16. 16.
    Leland, P., Sigler, C. I., Danforth, H. D., and Hollingdale, M. R. (1984) Inhibition of Plasmodium berghei sporozoite invasion of cultured hepatoma cells. Trans. R. Soc. Trop. Med. Hyg. 78, 639–640.CrossRefPubMedGoogle Scholar
  17. 17.
    Mazier, D., Mellouk, S., Beaudoin, R. L., Texier, B., Druilhe, P., Hockmeyer, W. T., et al. (1986) Effect of antibodies to recombinant and synthetic peptides on Plasmodium falciparum sporozoites in vitro. Science 231, 156–159.Google Scholar
  18. 18.
    Zavala, F., Hollingdale, M. R., Schwartz, A. L., Nussenzweig, R. S., and Nussenzweig, V. (1985) Immunoradiometric assay to measure the in vitro penetration of sporozoites of malaria parasites into hepatoma cells. J. Immunol. 134, 1202–1205.PubMedGoogle Scholar
  19. 19.
    Rénia, L., Miltgen, F., Charoenvit, Y., Ponnudurai, T. V., Verhave, J. P., Collins, W. E., and Mazier, D. (1988) Malaria sporozoite penetration: a new approach by double staining. J. Immunol. Methods, 112, 201–205.CrossRefPubMedGoogle Scholar
  20. 20.
    Nudelman, S., Rénia, L., Charoenvit, Y., Yuan, L., Miltgen, F., Beaudoin, R. L., and Mazier, D. (1989) Dual action of anti-sporozoite antibodies in vitro. J. Immunol. 143, 996–1000.Google Scholar
  21. 21.
    Frevert, U., Sinnis, P., Esko, J. D., and Nussenzweig, V. (1996) Cell surface glycosaminoglycans are not obligatory for Plasmodium berghei sporozoite invasion in vitro. Mol. Biochem. Parasitol. 76, 257–266.CrossRefPubMedGoogle Scholar
  22. 22.
    Sinnis, P. (1998) An immunoradiometric assay for the quantification of Plasmodium sporozoite invasion of HepG2 cells. J. Immunol. Methods 221, 17–23.CrossRefPubMedGoogle Scholar
  23. 23.
    Charoenvit, Y., Leef, M. F., Yuan, L. F., Sedegah, M., and Beaudoin, R. L. (1987) Characterization of Plasmodium yoelii monoclonal antibodies directed against stage-specific sporozoite antigens.Infect. Immun. 55, 604–608.PubMedGoogle Scholar
  24. 24.
    Millet, P., Collins, W. E., Herman, L., and Cochrane, A. H. (1989) Plasmodium vivax:In vitro development of exoerythrocytic stages in squirrel monkey hepatocytes and inhibition by an anti-P. cynomolgi monoclonal antibody. Exp. Parasitol. 69, 91–93.CrossRefPubMedGoogle Scholar
  25. 25.
    Millet, P., Kamboj, K. K., Cochrane, A. H., Collins, W. E., Broderson, J. R., Brown, B. G., et al.(1989) In-vitro exoerythrocytic development of Plasmodium cynomolgi bastianellii: inhibitory activity of monoclonal antibodies against sporozoites of different P. cynomolgi strains and of P.knowlesi. Parasite Immunol. 11, 223–230.CrossRefPubMedGoogle Scholar
  26. 26.
    Millet, P., Collins, W. E., Broderson, J. R., Bathurst, I., Nardin, E. H., and Nussenzweig, R. S.(1991) Inhibitory activity against Plasmodium vivax sporozoites induced by plasma from Saimiri monkeys immunized with circumsporozoite recombinant proteins or irradiated sporozoites. Am. J.Trop. Med. Hyg. 45, 44–48.PubMedGoogle Scholar
  27. 27.
    Etlinger, H. M., Rénia, L., Matile, H., Manneberg, M., Mazier, D., Trzeciak, A., and Gillessen, D.(1991) Antibody response to a synthetic peptide-based malaria vaccine candidate: influence of sequence variants of the peptide. Eur. J. Immunol. 21, 1505–1511.CrossRefPubMedGoogle Scholar
  28. 28.
    Fidock, D. A., Pasquetto, V., Gras, H., Badell, E., Eling, W. M. C., Ballou, W. R., et al. (1997) Plasmodium falciparum sporozoite invasion is inhibited by naturally acquired or experimentally induced polyclonal antibodies to the STARP antigen. Eur. J. Immunol. 27, 2502–2513.CrossRefPubMedGoogle Scholar
  29. 29.
    Hollingdale, M. R., Ballou, W. R., Aley, S. B., Young, J. F., Pancake, S., Miller, L. H., et al. (1987) Plasmodiumfalciparum: elicitation by peptides and recombinant circumsporozoite proteins of circulating mouse antibodies inhibiting sporozoite invasion of hepatoma cells. Exp. Parasitol. 63, 345–351.CrossRefPubMedGoogle Scholar
  30. 30.
    Moelans, I. I., Cohen, J., Marchand, M., Molitor, C., de Wilde, P., Van pelt, J. F., et al. (1995) Induction of Plasmodium falciparum sporozoite-neutralizing antibodies upon vaccination with recombinant Pfs16 vaccinia virus and/or recombinant Pfs16 protein produced in yeast. Mol.Biochem. Parasitol. 72, 179–192.CrossRefPubMedGoogle Scholar
  31. 31.
    Hollingdale, M. R., Appiah, A., Leland, P., Do Rosario, V. E., Mazier, D., Pied, S., et al. (1990) Activity of human volunteer sera to candidate Plasmodium falciparum circumsporozoite vaccines in the inhibition of sporozoite invasion (ISI) of human hepatoma cells and hepatocytes assays. Trans.R. Soc. Trop. Med. Hyg. 84, 325–329.CrossRefPubMedGoogle Scholar
  32. 32.
    Fries, L. F., Gordon, D. M., Richards, R. L., Egan, J. E., Hollingdale, M. R., Gross, M., et al. (1992) Liposomal malaria vaccine in humans: a safe and potent adjuvant strategy. Proc. Natl. Acad. Sci.USA 89, 358–362.CrossRefPubMedGoogle Scholar
  33. 33.
    Egan, J. E., Hoffman, S. L., Haynes, J. D., Sadoff, J. C., Schneider, I., Grau, G. E., et al. (1993) Humoral Immune Responses in Volunteers Immunized with Irradiated Plasmodium falciparum Sporozoites. Am. J. Trop. Med. Hyg. 49, 166–173.PubMedGoogle Scholar
  34. 34.
    Brown, A. E., Singharaj, P., Webster, H. K., Pipithkul, J., Gordon, D. M., Boslego, J. W., et al. (1994) Safety, Immunogenicity and Limited Efficacy Study of a Recombinant Plasmodium falciparum Circumsporozoite Vaccine in Thai Soldiers. Vaccine 12, 102–108.CrossRefPubMedGoogle Scholar
  35. 35.
    Mellouk, S., Mazier, D., Druilhe, P., Berbiguier, N., and Danis, M. (1986) In vitro and in vivo results suggest that antisporozoite antibodies do not totally block Plasmodium falciparum sporozoite infectivity. N. Engl. J. Med. 315, 648.PubMedGoogle Scholar
  36. 36.
    Mellouk, S., Lunel, F., Sedegah, M., Beaudoin, R. L., and Druilhe, P. (1990) Protection against malaria induced by irradiated sporozoites. Lancet 335, 721.CrossRefPubMedGoogle Scholar
  37. 37.
    Charoenvit, Y., Mellouk, S., Cole, C., Bechara, R., Leef, M. F., Sedegah, M., et al. (1991) Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites. J. Immunol. 146, 1020–1025.PubMedGoogle Scholar
  38. 38.
    Rénia, L., Mattei, D., Goma, J., Pied, S., Dubois, P., Miltgen, F., Nussler, A., et al. (1990) A malaria heat shock like protein epitope expressed on the infected hepatocyte surface is the target of antibody-dependent cell-mediated cytotoxic mechanisms by non-parechymal liver cells. Eur. J.Immunol. 20, 1445–1449.CrossRefPubMedGoogle Scholar
  39. 39.
    Nüssler, A., Pied, S., Pontet, M., Miltgen, F., Rénia, L., Gentilini, M., et al. (1991) Inflammatory status and pre-erythrocytic stages of malaria. Role of the C-reactive protein. Exp. Parasitol. 72, 1–7.CrossRefPubMedGoogle Scholar
  40. 40.
    Mellouk, S., Berbiguier, N., Druilhe, P., Sedegah, M., Galey, B., Yuan, L., et al. (1990) Evaluation of an in vitro assay aimed at measuring protective antibodies against sporozoites. Bull. World Hlth.Organ, 68 Suppl, 52–59.Google Scholar
  41. 41.
    Brahimi, K., Perignon, J. L., Bossus, M., Gras, H., Tartar, A., and Druilhe, P. (1993) Fast immunopurification of small amounts of specific antibodies on peptides bound to ELISA plates. J.Immunol. Methods 162, 69–75.CrossRefPubMedGoogle Scholar
  42. 42.
    Del Giudice, G., Tougne, C., Rénia, L., Ponnudurai, T., Corradin, G., Pessi, A., et al. (1991) Characterization of murine monoclonal antibodies against a repetitive synthetic peptide from the circumsporozoite protein of the human malaria parasite, Plasmodium falciparum. Mol. Immunol. 28, 1003–1009.Google Scholar
  43. 43.
    Pied, S., Nussler, A., Pontet, M., Miltgen, F., Matile, H., Lambert, P. H., and Mazier, D. (1989) Creactive protein protects against pre-erythrocytic stages of malaria. Infect. Immun. 57, 278–282.PubMedGoogle Scholar
  44. 44.
    Pied, S., Tabone, M. D., Chatellier, G., Marussig, M., Jardel, C., Nosten, F., and Mazier, D. (1995) Non specific resistance against malaria pre-erythrocytic stages: involvement of acute phase proteins. Parasite 2, 263–268.PubMedGoogle Scholar
  45. 45.
    Boulanger, N., Matile, H., and Betschart, B. (1988) Formation of the circumsporozoite protein of Plasmodium falciparum in Anopheles stephensi. Acta Tropica 45, 55–65.Google Scholar
  46. 46.
    Verhave, J. P., Leeuwenberg, A. D., Ponnudurai, T., Meuwissen, J. H., and van Druten, J. A. (1988) The biotin-streptavidin system in a two-site ELISA for the detection of plasmodial sporozoite antigen in mosquitoes. Parasite Immunol. 10, 17–31.CrossRefPubMedGoogle Scholar
  47. 47.
    Wirtz, R. A., Zavala, F., Charoenvit, Y., Campbell, G. H., Burkot, T. R., Schneider, I., et al. (1987) Comparative testing of monoclonal antibodies against Plasmodium falciparum sporozoites for ELISA development. Bull. World Hlth. Organ. 65, 39–45.Google Scholar
  48. 48.
    Yoshida, N., Nussenzweig, R. S., Potocnjak, P., Nussenzweig, V., and Aikawa, M. (1980) Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207, 71–73.CrossRefPubMedGoogle Scholar
  49. 49.
    Fidock, D. A., Bottius, E., Brahimi, K., Moelans, I. I., Aikawa, M., Konings, R. N., et al. (1994) Cloning and characterization of a novel Plasmodium falciparum sporozoite surface antigen, STARP.Mol. Biochem. Parasitol. 64, 219–232.CrossRefPubMedGoogle Scholar
  50. 50.
    Bottius, E., BenMohamed, L., Brahimi, K., Gras, H., Lepers, J. P., Raharimalala, L., et al. (1996) A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines maj or B, T helper, and CTL epitopes. J. Immunol. 156, 2874–2884.PubMedGoogle Scholar
  51. 51.
    Robson, K. J. H., Hall, J. R. S., Jennings, M. W., Harris, T. J. R., Marsh, K., Newbold, C. I., et al. (1988) A highly conserved amino-acid sequence in thrombospondin, properdin and in proteins from sporozoites and blood stages of a human malaria parasite. Nature 335, 79–82.CrossRefPubMedGoogle Scholar
  52. 52.
    Rogers, W. O., Malik, A., Mellouk, S., Nakamura, K., Rogers, M. D., Szarfman, A., et al. (1992) Characterization of Plasmodium falciparum sporozoite surface protein 2. Proc. Natl. Acad. Sci.USA 89, 9176–9180.CrossRefPubMedGoogle Scholar
  53. 53.
    Rogers, W. O., Rogers, M. D., Hedstrom, R. C., and Hoffman, S. L. (1992) Characterization of the gene encoding sporozoite surface protein 2, a protective Plasmodium yoelii sporozoite antigen. Mol.Biochem. Parasitol. 53, 45–51.CrossRefPubMedGoogle Scholar
  54. 54.
    Charoenvit, Y., Fallarme, V., Rogers, W. O., Sacci, J. B., Jr., Kaur, M., Aguiar, J. C., et al. (1997) Development of two monoclonal antibodies against Plasmodiumfalciparum sporozoite surface protein 2 and mapping of B-cell epitopes. Infect. Immun. 65, 3430–3437.PubMedGoogle Scholar
  55. 55.
    Lakowitz, J. R. and Weber, G. (1973) Quenching of fluorescence by oxygen: a probe for structural fluctuation in macromolecules. Biochemistry 21, 4161–4167.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Laurent Rénia
    • 1
  • Ana Margarida Vigário
    • 1
  • Elodie Belnoue
    • 1
  1. 1.Département d’Immunologie, Institut Cochin, INSERM/CNRSUniversité René Déscartes; Hôpital CochinParisFrance

Personalised recommendations