Immunization with attenuated Plasmodium falciparum sporozoites (PfSPZs) has been shown to be protective against malaria, but the features of the antibody response induced by this treatment remain unclear. To investigate this response in detail, we isolated IgM and IgG monoclonal antibodies from Tanzanian volunteers who were immunized with repeated injection of Sanaria PfSPZ Vaccine and who were found to be protected from controlled human malaria infection with infectious homologous PfSPZs. All isolated IgG monoclonal antibodies bound to P. falciparum circumsporozoite protein (PfCSP) and recognized distinct epitopes in its N terminus, NANP-repeat region, and C terminus. Strikingly, the most effective antibodies, as determined in a humanized mouse model, bound not only to the repeat region, but also to a minimal peptide at the PfCSP N-terminal junction that is not in the RTS,S vaccine. These dual-specific antibodies were isolated from different donors and were encoded by VH3-30 or VH3-33 alleles that encode tryptophan or arginine at position 52. Using structural and mutational data, we describe the elements required for germline recognition and affinity maturation. Our study provides potent neutralizing antibodies and relevant information for lineage-targeted vaccine design and immunization strategies.

  • Subscribe to Nature Medicine for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


Primary accessions


Protein Data Bank


  1. 1.

    World Health Organization. World malaria report 2016. (World Health Organization, 2017).

  2. 2.

    et al. Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science 237, 639–642 (1987).

  3. 3.

    et al. An intensive longitudinal cohort study of Malian children and adults reveals no evidence of acquired immunity to Plasmodium falciparum infection. Clin. Infect. Dis. 57, 40–47 (2013).

  4. 4.

    , & The RTS,S malaria vaccine. Vaccine 28, 4880–4894 (2010).

  5. 5.

    , & Antibody and B cell responses to Plasmodium sporozoites. Front. Microbiol. 5, 625 (2014).

  6. 6.

    et al. Protection against malaria at 1 year and immune correlates following PfSPZ vaccination. Nat. Med. 22, 614–623 (2016).

  7. 7.

    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).

  8. 8.

    et al. Malaria circumsporozoite protein binds to heparan sulfate proteoglycans associated with the surface membrane of hepatocytes. J. Exp. Med. 177, 1287–1298 (1993).

  9. 9.

    , , & The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion. J. Exp. Med. 201, 27–33 (2005).

  10. 10.

    RTS,S Clinical Trials Partnership. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 386, 31–45 (2015).

  11. 11.

    et al. Heparan sulfate proteoglycans provide a signal to Plasmodium sporozoites to stop migrating and productively invade host cells. Cell Host Microbe 2, 316–327 (2007).

  12. 12.

    et al. The malaria circumsporozoite protein has two functional domains, each with distinct roles as sporozoites journey from mosquito to mammalian host. J. Exp. Med. 208, 341–356 (2011).

  13. 13.

    et al. Four-year efficacy of RTS,S/AS01E and its interaction with malaria exposure. N. Engl. J. Med. 368, 1111–1120 (2013).

  14. 14.

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

  15. 15.

    , , & Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature 216, 160–162 (1967).

  16. 16.

    , , & Immunization of man against sporozite-induced falciparum malaria. Am. J. Med. Sci. 266, 169–177 (1973).

  17. 17.

    , , , & Letter: sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans. R. Soc. Trop. Med. Hyg. 68, 258–259 (1974).

  18. 18.

    et al. Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J. Infect. Dis. 185, 1155–1164 (2002).

  19. 19.

    et al. Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria. Hum. Vaccin. 6, 97–106 (2010).

  20. 20.

    et al. Protection against malaria by intravenous immunization with a nonreplicating sporozoite vaccine. Science 341, 1359–1365 (2013).

  21. 21.

    et al. Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 542, 445–449 (2017).

  22. 22.

    et al. Safety and efficacy of PfSPZ Vaccine against Plasmodium falciparum via direct venous inoculation in healthy malaria-exposed adults in Mali: a randomised, double-blind phase 1 trial. Lancet Infect. Dis. 17, 498–509 (2017).

  23. 23.

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

  24. 24.

    et al. The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444, 937–940 (2006).

  25. 25.

    et al. Natural parasite exposure induces protective human anti-malarial antibodies. Immunity 47, 1197–1209.e10 (2017).

  26. 26.

    , , & Conformational preferences of synthetic peptides derived from the immunodominant site of the circumsporozoite protein of Plasmodium falciparum by 1H NMR. Biochemistry 29, 7828–7837 (1990).

  27. 27.

    , , & Crystal structure of an NPNA-repeat motif from the circumsporozoite protein of the malaria parasite Plasmodium falciparum. Chem. Commun. (Camb.) 365, 174–176 (2006).

  28. 28.

    et al. Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein. Proc. Natl. Acad. Sci. USA 114, E10438–E10445 (2017).

  29. 29.

    et al. T-dependent B cell responses to Plasmodium induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein. PLoS Pathog. 13, e1006469 (2017).

  30. 30.

    , , , & Identification of five different IGHV gene families in owl monkeys (Aotus nancymaae). Tissue Antigens 66, 640–649 (2005).

  31. 31.

    et al. Somatically hypermutated Plasmodium-specific IgM+ memory B cells are rapid, plastic, early responders upon malaria rechallenge. Immunity 45, 402–414 (2016).

  32. 32.

    , & Human marginal zone B cells. Annu. Rev. Immunol. 27, 267–285 (2009).

  33. 33.

    et al. Total and putative surface proteomics of malaria parasite salivary gland sporozoites. Mol. Cell. Proteomics 12, 1127–1143 (2013).

  34. 34.

    et al. Interrogating the Plasmodium sporozoite surface: identification of surface-exposed proteins and demonstration of glycosylation on CSP and TRAP by mass spectrometry-based proteomics. PLoS Pathog. 12, e1005606 (2016).

  35. 35.

    et al. An immunologically cryptic epitope of Plasmodium falciparum circumsporozoite protein facilitates liver cell recognition and induces protective antibodies that block liver cell invasion. J. Biol. Chem. 280, 20524–20529 (2005).

  36. 36.

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

  37. 37.

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

  38. 38.

    et al. Versatile virus-like particle carrier for epitope based vaccines. PLoS One 5, e9809 (2010).

  39. 39.

    et al. Corrigendum: Design of a hyperstable 60-subuni 60-subunit protein icosahedron. Nature 540, 150 (2016).

  40. 40.

    et al. Rapid development of broadly influenza neutralizing antibodies through redundant mutations. Nature 516, 418–422 (2014).

  41. 41.

    et al. Vaccine-induced antibodies that neutralize group 1 and group 2 influenza A viruses. Cell 166, 609–623 (2016).

  42. 42.

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

  43. 43.

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

  44. 44.

    et al. Humoral protection against mosquito bite-transmitted Plasmodium falciparum infection in humanized mice. NPJ Vaccines 2, 27 (2017).

  45. 45.

    et al. Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein. Mol. Immunol. 32, 1301–1309 (1995).

  46. 46.

    et al. A LAIR1 insertion generates broadly reactive antibodies against malaria variant antigens. Nature 529, 105–109 (2016).

  47. 47.

    et al. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat. Med. 10, 871–875 (2004).

  48. 48.

    , , & Development of a quantitative flow cytometry–based assay to assess infection by Plasmodium falciparum sporozoites. Mol. Biochem. Parasitol. 183, 100–103 (2012).

  49. 49.

    et al. Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. J. Immunol. Methods 329, 112–124 (2008).

  50. 50.

    et al. IMGT, the international immunogenetics information system. Nucleic Acids Res. 37, D1006–D1012 (2009).

  51. 51.

    et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38, W695–9 (2010).

  52. 52.

    Reconstructing a B-cell clonal lineage. I. Statistical inference of unobserved ancestors. F1000Res. 2, 103 (2013).

  53. 53.

    et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469–476 (2013).

  54. 54.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  55. 55.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

  56. 56.

    et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–8 (2014).

  57. 57.

    et al. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4, 1–13 (2009).

  58. 58.

    , , & The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201 (2006).

  59. 59.

    , , & PIGSPro: prediction of immunoglobulin structures v2. Nucleic Acids Res. 45 W1, W17–W23 (2017).

  60. 60.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  61. 61.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  62. 62.

    The molecular surface package. J. Mol. Graph. 11, 139–141 (1993).

  63. 63.

    & Side-chain torsional potentials: effect of dipeptide, protein, and solvent environment. Biochemistry 18, 1256–1268 (1979).

  64. 64.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

Download references


We would like to thank M. Nussenzweig (Rockefeller University) and H. Wardemann (German Cancer Research Center) for providing reagents for antibody cloning and expression. This work was supported in part by the Swiss Vaccine Research Institute, by the European Research Council (grant no. 670955 BROADimmune) and by the Fondazione Aldo e Cele Daccò. J.T. is funded by the Wellcome Trust (grant no. 204689/Z/16/Z). B.S. is funded by the National Institutes of Health (grant no. F32 AI 114113). The authors would like to thank first and foremost the study volunteers for their participation in the study. We also thank the entire study team at the Bagamoyo branch of the Ifakara Health Institute and the manufacturing, quality control, regulatory and clinical teams at Sanaria, Inc. for their contributions to the conduct of the trial. We would like to thank M. Tanner (former director of the Swiss Tropical and Public Health Institute, Basel) for his vision and support of the development of the clinical trial platform enabling whole sporozoite–based malaria vaccine trials in Bagamoyo, Tanzania. D.O. and I.A.W. are funded by PATH's Malaria Vaccine Initiative under a collaborative agreement with The Scripps Research Institute and by the Bill and Melinda Gates Foundation. A.L. and F.S. are supported by the Helmut Horten Foundation. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under contract no. DE-AC02-05CH11231. The Tanzanian Commission on Science and Technology (COSTECH), the Ifakara Health Institute, and the Swiss Tropical Public Health Institute provided funding for the clinical trial. The functional assays were supported by the Bill and Melinda Gates foundation (Investment ID: 24922). The development, manufacturing, and quality control release and stability studies of PfSPZ Vaccine and PfSPZ Challenge were supported in part by National Institute of Allergy and Infectious Diseases (NIAID) Small Business Innovation Research grant 5R44AI055229. Sanaria supported transport of PfSPZ Vaccine and PfSPZ Challenge to the study site and syringe preparation.

Author information

Author notes

    • Joshua Tan
    • , Brandon K Sack
    • , David Oyen
    • , Isabelle Zenklusen
    •  & Luca Piccoli

    These authors contributed equally to this work.

    • Claudia Daubenberger
    • , Ian A Wilson
    •  & Antonio Lanzavecchia

    These authors jointly directed this work.


  1. Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland.

    • Joshua Tan
    • , Luca Piccoli
    • , Sonia Barbieri
    • , Mathilde Foglierini
    • , Chiara Silacci Fregni
    • , Jessica Marcandalli
    • , Laurent Perez
    • , Luca Varani
    • , Federica Sallusto
    •  & Antonio Lanzavecchia
  2. Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.

    • Joshua Tan
  3. Center for Infectious Disease Research, Seattle, Washington, USA.

    • Brandon K Sack
    •  & Stefan H I Kappe
  4. Department of Integrative Structural and Computational Biology, Scripps Research Institute, La Jolla, California, USA.

    • David Oyen
    •  & Ian A Wilson
  5. Faculty of Science, University of Basel, Basel, Switzerland.

    • Isabelle Zenklusen
    •  & Claudia Daubenberger
  6. Swiss Tropical and Public Health Institute, Clinical Immunology Unit, Basel, Switzerland.

    • Isabelle Zenklusen
    •  & Claudia Daubenberger
  7. Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.

    • Mathilde Foglierini
  8. Ifakara Health Institute, Bagamoyo Clinical Trial Unit, Bagamoyo, Tanzania.

    • Said Jongo
    •  & Salim Abdulla
  9. Biochemistry Department, University of Lausanne, Epalinges, Switzerland.

    • Giampietro Corradin
  10. Institute of Microbiology, ETH Zurich, Zurich, Switzerland.

    • Federica Sallusto
    •  & Antonio Lanzavecchia
  11. Sanaria Inc., Rockville, Maryland, USA.

    • Betty Kim Lee Sim
    •  & Stephen L Hoffman
  12. Skaggs Institute for Chemical Biology, Scripps Research Institute, La Jolla, California, USA.

    • Ian A Wilson


  1. Search for Joshua Tan in:

  2. Search for Brandon K Sack in:

  3. Search for David Oyen in:

  4. Search for Isabelle Zenklusen in:

  5. Search for Luca Piccoli in:

  6. Search for Sonia Barbieri in:

  7. Search for Mathilde Foglierini in:

  8. Search for Chiara Silacci Fregni in:

  9. Search for Jessica Marcandalli in:

  10. Search for Said Jongo in:

  11. Search for Salim Abdulla in:

  12. Search for Laurent Perez in:

  13. Search for Giampietro Corradin in:

  14. Search for Luca Varani in:

  15. Search for Federica Sallusto in:

  16. Search for Betty Kim Lee Sim in:

  17. Search for Stephen L Hoffman in:

  18. Search for Stefan H I Kappe in:

  19. Search for Claudia Daubenberger in:

  20. Search for Ian A Wilson in:

  21. Search for Antonio Lanzavecchia in:


J.T. characterized monoclonal antibodies, analyzed the data and wrote the manuscript; B.K.S. performed in vivo assays, analyzed the data and wrote the manuscript; D.O. performed structural analysis, analyzed the data and wrote the manuscript; I.Z. collected samples, conducted in vitro assays, analyzed the data and wrote the manuscript; L. Piccoli characterized monoclonal antibodies, analyzed the data and wrote the manuscript; S.B. sequenced and expressed antibodies; M.F. performed bioinformatics analysis; C.S.F. immortalized memory B cells; J.M and L. Perez immunized mice; S.J. supervised cohorts; S.A. oversaw the clinical trial and provided PBMCs to the laboratory team; G.C. provided PfCSP peptides; L.V. designed peptide and antibody mutants; F.S. and S.H.I.K. provided supervision; S.L.H. and B.K.L.S. produced PfSPZ Vaccine and PfCSP, prepared the syringes used to immunize, and provided PfSPZ for antibody assays, including screening of monoclonal antibodies; C.D. handled cohorts and provided supervision; I.A.W. supervised structural analysis and wrote the manuscript; A.L. provided overall supervision, analyzed the data and wrote the manuscript.

Competing interests

A.L. is the scientific founder and shareholder of Humabs BioMed. F.S. is a shareholder of Humabs BioMed. B.K.L.S. and S.L.H. are salaried employees of Sanaria Inc., the developer and owner of the PfSPZ Vaccine and PfSPZ Challenge and the sponsor of the clinical trials. In addition, B.K.L.S. and S.L.H. have a financial interest in Sanaria Inc.

Corresponding author

Correspondence to Antonio Lanzavecchia.

Supplementary information

PDF files

  1. 1.

    Supplementary Figures & Tables

    Supplementary Figures 1–9 & Supplementary Table 1–2

  2. 2.

    Life Sciences Reporting Summary

About this article

Publication history






Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.