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Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin


The circumsporozoite protein (CSP) is the major surface protein of malaria sporozoites (SPZs), the motile and invasive parasite stage inoculated in the host skin by infected mosquitoes. Antibodies against the central CSP repeats of different plasmodial species are known to block SPZ infectivity1,2,3,4,5, but the precise mechanism by which these effectors operate is not completely understood. Here, using a rodent Plasmodium yoelii malaria model, we show that sterile protection mediated by anti-P. yoelii CSP humoral immunity depends on the parasite inoculation into the host skin, where antibodies inhibit motility and kill P. yoelii SPZs via a characteristic ‘dotty death’ phenotype. Passive transfer of an anti-repeat monoclonal antibody (mAb) recapitulates the skin inoculation-dependent protection, in a complement- and Fc receptor γ-independent manner. This purified mAb also decreases motility and, notably, induces the dotty death of P. yoelii SPZs in vitro. Cytotoxicity is species-transcendent since cognate anti-CSP repeat mAbs also kill Plasmodium berghei and Plasmodium falciparum SPZs. mAb cytotoxicity requires the actomyosin motor-dependent translocation and stripping of the protective CSP surface coat, rendering the parasite membrane susceptible to the SPZ pore-forming-like protein secreted to wound and traverse the host cell membrane6. The loss of SPZ fitness caused by anti-P. yoelii CSP repeat antibodies is thus a dynamic process initiated in the host skin where SPZs either stop moving7, or migrate and traverse cells to progress through the host tissues7,8,9 at the eventual expense of their own life.

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Fig. 1: Sterilizing anti-P. yoelii CSP humoral immunity is dependent on the host skin inoculation with SPZs.
Fig. 2: Decrease of motility and killing of parasites in the skin of CSP-immunized mice.
Fig. 3: Complement and FcRγ-independent cytotoxic antibody sterilizes SPZ infection.
Fig. 4: Motility, secretion and SPZ-specific wounding proteins play a critical role in the death mediated by the cytotoxic antibody.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.


  1. 1.

    Yoshida, N., Nussenzweig, R. S., Potocnjak, P., Nussenzweig, V. & Aikawa, M. Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207, 71–73 (1980).

    CAS  Article  Google Scholar 

  2. 2.

    Nardin, E. H. et al. Circumsporozoite proteins of human malaria parasites Plasmodium falciparum and Plasmodium vivax. J. Exp. Med. 156, 20–30 (1982).

    CAS  Article  Google Scholar 

  3. 3.

    Cochrane, A. H., Santoro, F., Nussenzweig, V., Gwadz, R. W. & Nussenzweig, R. S. Monoclonal antibodies identify the protective antigens of sporozoites of Plasmodium knowlesi. Proc. Natl Acad. Sci. USA 79, 5651–5655 (1982).

    CAS  Article  Google Scholar 

  4. 4.

    Charoenvit, Y. et al. Inability of malaria vaccine to induce antibodies to a protective epitope within its sequence. Science 251, 668–671 (1991).

    CAS  Article  Google Scholar 

  5. 5.

    Charoenvit, Y. et al. Monoclonal, but not polyclonal, antibodies protect against Plasmodium yoelii sporozoites. J. Immunol. 146, 1020–1025 (1991).

    CAS  PubMed  Google Scholar 

  6. 6.

    Ishino, T., Chinzei, Y. & Yuda, M. A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell Microbiol. 7, 199–208 (2005).

    CAS  Article  Google Scholar 

  7. 7.

    Vanderberg, J. P. & Frevert, U. Intravital microscopy demonstrating antibody-mediated immobilisation of Plasmodium berghei sporozoites injected into skin by mosquitoes. Int. J. Parasitol. 34, 991–996 (2004).

    Article  Google Scholar 

  8. 8.

    Amino, R. et al. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nat. Med. 12, 220–224 (2006).

    CAS  Article  Google Scholar 

  9. 9.

    Amino, R. et al. Host cell traversal is important for progression of the malaria parasite through the dermis to the liver. Cell Host Microbe 3, 88–96 (2008).

    CAS  Article  Google Scholar 

  10. 10.

    Ménard, R. et al. Circumsporozoite protein is required for development of malaria sporozoites in mosquitoes. Nature 385, 336–340 (1997).

    Article  Google Scholar 

  11. 11.

    Pancake, S. J., Holt, G. D., Mellouk, S. & Hoffman, S. L. Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates. J. Cell Biol. 117, 1351–1357 (1992).

    CAS  Article  Google Scholar 

  12. 12.

    Sidjanski, S. P., Vanderberg, J. P. & Sinnis, P. Anopheles stephensi salivary glands bear receptors for region I of the circumsporozoite protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 90, 33–41 (1997).

    CAS  Article  Google Scholar 

  13. 13.

    Cerami, C. et al. The basolateral domain of the hepatocyte plasma membrane bears receptors for the circumsporozoite protein of Plasmodium falciparum sporozoites. Cell 70, 1021–1033 (1992).

    CAS  Article  Google Scholar 

  14. 14.

    Tewari, R., Spaccapelo, R., Bistoni, F., Holder, A. A. & Crisanti, A. Function of region I and II adhesive motifs of Plasmodium falciparum circumsporozoite protein in sporozoite motility and infectivity. J. Biol. Chem. 277, 47613–47618 (2002).

    CAS  Article  Google Scholar 

  15. 15.

    Gilson, P. R. et al. Identification and stoichiometry of glycosylphosphatidylinositol-anchored membrane proteins of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 5, 1286–1299 (2006).

    CAS  Article  Google Scholar 

  16. 16.

    Olotu, A. et al. Seven-year efficacy of RTS,S/AS01 malaria vaccine among young African children. N. Engl. J. Med. 374, 2519–2529 (2016).

    CAS  Article  Google Scholar 

  17. 17.

    White, M. T. et al. The relationship between RTS,S vaccine-induced antibodies, CD4+ T cell responses and protection against Plasmodium falciparum infection. PLoS ONE 8, e61395 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Stewart, M. J., Nawrot, R. J., Schulman, S. & Vanderberg, J. P. Plasmodium berghei sporozoite invasion is blocked in vitro by sporozoite-immobilizing antibodies. Infect. Immun. 51, 859–864 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Hollingdale, M. R., Zavala, F., Nussenzweig, R. S. & Nussenzweig, V. Antibodies to the protective antigen of Plasmodium berghei sporozoites prevent entry into cultured cells. J. Immunol. 128, 1929–1930 (1982).

    CAS  PubMed  Google Scholar 

  20. 20.

    Hollingdale, M. R., Nardin, E. H., Tharavanij, S., Schwartz, A. L. & Nussenzweig, R. S. 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 (1984).

    CAS  PubMed  Google Scholar 

  21. 21.

    Potocnjak, P., Yoshida, N., Nussenzweig, R. S. & Nussenzweig, V. Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. J. Exp. Med. 151, 1504–1513 (1980).

    CAS  Article  Google Scholar 

  22. 22.

    Egan, J. E. et al. Efficacy of murine malaria sporozoite vaccines: implications for human vaccine development. Science 236, 453–456 (1987).

    CAS  Article  Google Scholar 

  23. 23.

    Kisalu, N. K. et al. A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite. Nat. Med. 24, 408–416 (2018).

    CAS  Article  Google Scholar 

  24. 24.

    Sack, B. K. et al. Model for in vivo assessment of humoral protection against malaria sporozoite challenge by passive transfer of monoclonal antibodies and immune serum. Infect. Immun. 82, 808–817 (2014).

    Article  Google Scholar 

  25. 25.

    Sedegah, M. et al. Evaluation of vaccines designed to induce protective cellular immunity against the Plasmodium yoelii circumsporozoite protein: vaccinia, pseudorabies, and salmonella transformed with circumsporozoite gene. Bull. World Health Organ. 68, 9–114 (1990).

    Google Scholar 

  26. 26.

    Collins, W. E. et al. Immunization of Saimiri sciureus boliviensis with recombinant vaccines based on the circumsporozoite protein of Plasmodium vivax. Am. J. Trop. Med. Hyg. 40, 455–464 (1989).

    CAS  Article  Google Scholar 

  27. 27.

    Lal, A. A. et al. In vivo testing of subunit vaccines against malaria sporozoites using a rodent system. Proc. Natl Acad. Sci. USA 84, 8647–8651 (1987).

    CAS  Article  Google Scholar 

  28. 28.

    Belmonte, M. et al. The infectivity of Plasmodium yoelii in different strains of mice. J. Parasitol. 89, 602–603 (2003).

    CAS  Article  Google Scholar 

  29. 29.

    Gueirard, P. et al. Development of the malaria parasite in the skin of the mammalian host. Proc. Natl Acad. Sci. USA 107, 18640–18645 (2010).

    CAS  Article  Google Scholar 

  30. 30.

    Bongfen, S. E. et al. The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity. Vaccine 27, 328–335 (2009).

    CAS  Article  Google Scholar 

  31. 31.

    Herrera, R. et al. Reversible conformational change in the Plasmodium falciparum circumsporozoite protein masks its adhesion domains. Infect. Immun. 83, 3771–3780 (2015).

    CAS  Article  Google Scholar 

  32. 32.

    Espinosa, D. A. et al. Proteolytic cleavage of the Plasmodium falciparum circumsporozoite protein is a target of protective antibodies. J. Infect. Dis. 212, 1111–1119 (2015).

    CAS  Article  Google Scholar 

  33. 33.

    Noris, M. & Remuzzi, G. Overview of complement activation and regulation. Semin. Nephrol. 33, 479–492 (2013).

    CAS  Article  Google Scholar 

  34. 34.

    Nimmerjahn, F. & Ravetch, J. V. Fcγ receptors: old friends and new family members. Immunity 24, 19–28 (2006).

    CAS  Article  Google Scholar 

  35. 35.

    Persson, C. et al. Cutting edge: a new tool to evaluate human pre-erythrocytic malaria vaccines: rodent parasites bearing a hybrid Plasmodium falciparum circumsporozoite protein. J. Immunol. 169, 6681–6685 (2002).

    CAS  Article  Google Scholar 

  36. 36.

    Vanderberg, J. P. Studies on the motility of Plasmodium sporozoites. J. Protozool. 21, 527–537 (1974).

    CAS  Article  Google Scholar 

  37. 37.

    Stewart, M. J. & Vanderberg, J. P. Malaria sporozoites release circumsporozoite protein from their apical end and translocate it along their surface. J. Protozool. 38, 411–421 (1991).

    CAS  Article  Google Scholar 

  38. 38.

    Carey, A. F. et al. Calcium dynamics of Plasmodium berghei sporozoite motility. Cell Microbiol. 16, 768–783 (2014).

    CAS  Article  Google Scholar 

  39. 39.

    Risco-Castillo, V. et al. Malaria sporozoites traverse host cells within transient vacuoles. Cell Host Microbe 18, 593–603 (2015).

    CAS  Article  Google Scholar 

  40. 40.

    Ishino, T., Yano, K., Chinzei, Y. & Yuda, M. Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol. 2, E4 (2004).

    Article  Google Scholar 

  41. 41.

    Kariu, T., Ishino, T., Yano, K., Chinzei, Y. & Yuda, M. CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Mol. Microbiol. 59, 1369–1379 (2006).

    CAS  Article  Google Scholar 

  42. 42.

    Jimah, J. R. Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. eLife 5, e20621 (2016).

  43. 43.

    Fine, E., Aikawa, M., Cochrane, A. H. & Nussenzweig, R. S. Immuno-electron microscopic observations on Plasmodium knowlesi sporozoites: localization of protective antigen and its precursors. Am. J. Trop. Med. Hyg. 33, 220–226 (1984).

    CAS  Article  Google Scholar 

  44. 44.

    Posthuma, G. et al. Immunogold determination of Plasmodium falciparum circumsporozoite protein in Anopheles stephensi salivary gland cells. Eur. J. Cell Biol. 49, 66–72 (1989).

    CAS  PubMed  Google Scholar 

  45. 45.

    Wang, R. et al. Induction of protective polyclonal antibodies by immunization with a Plasmodium yoelii circumsporozoite protein multiple antigen peptide vaccine. J. Immunol. 154, 2784–2793 (1995).

    CAS  PubMed  Google Scholar 

  46. 46.

    Yilmaz, B. et al. Gut microbiota elicits a protective immune response against malaria transmission. Cell 159, 1277–1289 (2014).

    CAS  Article  Google Scholar 

  47. 47.

    Bouharoun-Tayoun, H., Oeuvray, C., Lunel, F. & Druilhe, P. Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages. J. Exp. Med. 182, 409–418 (1995).

    CAS  Article  Google Scholar 

  48. 48.

    Weinbaum, F. I., Evans, C. B. & Tigelaar, R. E. An in vitro assay for T cell immunity to malaria in mice. J. Immunol. 116, 1280–1283 (1976).

    CAS  PubMed  Google Scholar 

  49. 49.

    Ono, T., Tadakuma, T. & Rodriguez, A. Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle. Exp. Parasitol. 115, 310–313 (2007).

    CAS  Article  Google Scholar 

  50. 50.

    Manzoni, G. et al. A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites. Sci. Rep. 4, 4760 (2014).

    Article  Google Scholar 

  51. 51.

    Mwakingwe, A. et al. Noninvasive real-time monitoring of liver-stage development of bioluminescent Plasmodium parasites. J. Infect. Dis. 200, 1470–1478 (2009).

    CAS  Article  Google Scholar 

  52. 52.

    Ishino, T., Orito, Y., Chinzei, Y. & Yuda, M. A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell. Mol. Microbiol. 59, 1175–1184 (2006).

    CAS  Article  Google Scholar 

  53. 53.

    Sturm, A. et al. Alteration of the parasite plasma membrane and the parasitophorous vacuole membrane during exo-erythrocytic development of malaria parasites. Protist 160, 51–63 (2009).

    Article  Google Scholar 

  54. 54.

    Demarta-Gatsi, C. et al. Immunological memory to blood-stage malaria infection is controlled by the histamine releasing factor (HRF) of the parasite. Sci. Rep. 7, 9129 (2017).

    Article  Google Scholar 

  55. 55.

    Ponnudurai, T., Leeuwenberg, A. D. & Meuwissen, J. H. Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture. Trop. Geogr. Med. 33, 50–54 (1981).

    CAS  PubMed  Google Scholar 

  56. 56.

    Gu, H., Zou, Y. R. & Rajewsky, K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73, 1155–1164 (1993).

    CAS  Article  Google Scholar 

  57. 57.

    Wessels, M. R. et al. Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc. Natl Acad. Sci. USA 92, 11490–11494 (1995).

    CAS  Article  Google Scholar 

  58. 58.

    Takai, T., Li, M., Sylvestre, D. & Clynes, R. FcR γ chain deletion results in pleiotrophiceffector cell defects.Cell 76, 519–529 (1994).

    CAS  Article  Google Scholar 

  59. 59.

    Ponnudurai, T., Lensen, A. H. & Meuwissen, J. H. An automated large-scale culture system of Plasmodium falciparum using tangential flow filtration for medium change. Parasitology 87(Pt 3), 439–445 (1983).

    Article  Google Scholar 

  60. 60.

    Liu, S. et al. Removal of endotoxin from recombinant protein preparations. Clin. Biochem. 30, 455–463 (1997).

    CAS  Article  Google Scholar 

  61. 61.

    Foquet, L. et al. Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection. J. Clin. Invest. 124, 140–144 (2014).

    CAS  Article  Google Scholar 

  62. 62.

    Schmidt, N. W., Butler, N. S., Badovinac, V. P. & Harty, J. T. Extreme CD8 T cell requirements for anti-malarial liver-stage immunity following immunization with radiation attenuated sporozoites. PLoS Pathog. 6, e1000998 (2010).

    Article  Google Scholar 

  63. 63.

    Jaeger, B. N. et al. Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis. J. Exp. Med. 209, 565–580 (2012).

    CAS  Article  Google Scholar 

  64. 64.

    Amino, R. et al. Imaging malaria sporozoites in the dermis of the mammalian host. Nat. Protoc. 2, 1705–1712 (2007).

    CAS  Article  Google Scholar 

  65. 65.

    Köhler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497 (1975).

    Article  Google Scholar 

  66. 66.

    Eichinger, D. J., Arnot, D. E., Tam, J. P., Nussenzweig, V. & Enea, V. Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes. Mol. Cell. Biol. 6, 3965–3972 (1986).

    CAS  Article  Google Scholar 

  67. 67.

    Zavala, F. et al. Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria. Science 228, 1436–1440 (1985).

    CAS  Article  Google Scholar 

  68. 68.

    Prudêncio, M., Rodrigues, C. D., Ataíde , R. & Mota, M. M. Dissecting in vitro host cell infection by Plasmodium sporozoites using flow cytometry. Cell. Microbiol. 10, 218–224 (2008).

    PubMed  Google Scholar 

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We thank the team of the Centre of Production and Infection of Anopheles, Institut Pasteur, in particular M. Szatanik, C. Thouvenot, S. Golba, J. Pham and A. Lorthiois for providing mosquitoes and P. falciparum SPZs; the team of the Platform of Dynamic Imaging, Institut Pasteur, in particular Dr S. Shorte and M.-A. Nicola for the access to the confocal microscopes and IVIS system; Dr K. Kim from the Albert Einstein College of Medicine for providing the P. yoelii YM GFP-luciferase; Dr V. Nussenzweig from New York University for providing the P. berghei and falciparumnized P. berghei parasites; Dr M. Soares from the Instituto Gulbenkian for providing the JHT/ mice; Dr P.-M. Lledo and Dr P. Bruhns from the Institut Pasteur for providing, respectively, the C3/ and the FcRγ/ mice; the team of the Clinical Investigation and Access to BioResources, in particular M.-N. Ungeheuer for providing the erythrocytes for the P. falciparum culture; Dr P. Baldacci and Dr P. Formaglio for their critical reading of the manuscript. This work was supported by funds from Institut Pasteur, Paris, the French National Research Agency (grant no. ANR-14-CE16-Im3alaria), the French Government’s ‘Investissement d’Avenir’ program, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant no. ANR-10-LABX-62-IBEID) and ‘ParaFrap’ (grant no. ANR-11-LABX-0024), the São Paulo Research Foundation (FAPESP, grant no. 2014/50631-0), the National Council for Scientific and Technological Development (CNPq), the BNP Paribas CIB, the Portuguese Science and Technology Foundation (FCT, grant no. IF/00881/2012/CP0158) and the European Social Fund (Human Potential Operational Programme).

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R.A., S.B.B., E.A. and J.T. conceived and designed the experiments. E.A., J.T., S.T. and R.H.P performed the experiments. M.M.Y., S.D. and F.T. generated the hybridomas. O.S., T.I. and M.Y. contributed with the transgenic parasites. R.A., S.B.B., E.A., J.T., S.T. and R.H.P. analysed the data. R.A, E.A, J.T. and S.B.B. wrote the paper.

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Correspondence to Silvia Beatriz Boscardin or Rogerio Amino.

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Aliprandini, E., Tavares, J., Panatieri, R.H. et al. Cytotoxic anti-circumsporozoite antibodies target malaria sporozoites in the host skin. Nat Microbiol 3, 1224–1233 (2018).

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