Evolution of protective human antibodies against Plasmodium falciparum circumsporozoite protein repeat motifs

Abstract

The circumsporozoite protein of the human malaria parasite Plasmodium falciparum (PfCSP) is the main target of antibodies that prevent the infection and disease, as shown in animal models. However, the limited efficacy of the PfCSP-based vaccine RTS,S calls for a better understanding of the mechanisms driving the development of the most potent human PfCSP antibodies and identification of their target epitopes. By characterizing 200 human monoclonal PfCSP antibodies induced by sporozoite immunization, we establish that the most potent antibodies bind around a conserved (N/D)PNANPN(V/A) core. High antibody affinity to the core correlates with protection from parasitemia in mice and evolves around the recognition of NANP motifs. The data suggest that the rational design of a next-generation PfCSP vaccine that elicits high-affinity antibody responses against the core epitope will promote the induction of protective humoral immune responses.

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Fig. 1: Cross-reactivity and affinity profiles of human PfCSP-reactive antibodies.
Fig. 2: PfCSP peptide recognition by IGHV3-33-encoded PfCSP-reactive antibodies.
Fig. 3: Preferential recognition of different PfCSP peptides around the NANP core epitope by the non-IGHV3-33-encoded cross-reactive mAb 4493.
Fig. 4: Selection and maturation of anti-PfCSP cross-reactive antibody profiles over successive Pf exposures.
Fig. 5: Parasite-inhibitory activity of cross-reactive PfCSP antibodies with different binding profiles.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding authors under standard material transfer agreements. The crystal structures reported in this manuscript have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID codes 6O23, 6O24, 6O25, 6O26, 6O28, 6O29, 6O2A, 6O2B, 6O2C, 6ULE, 6ULF, 6VLN).

References

  1. 1.

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

    CAS  PubMed  Google Scholar 

  2. 2.

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

    PubMed  Google Scholar 

  3. 3.

    Coppi, A. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Dame, J. B. et al. Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science 225, 593–599 (1984).

    CAS  PubMed  Google Scholar 

  5. 5.

    Enea, V. et al. DNA cloning of Plasmodium falciparum circumsporozoite gene: amino acid sequence of repetitive epitope. Science 225, 628–630 (1984).

    CAS  Google Scholar 

  6. 6.

    Escalante, A. A. et al. A study of genetic diversity in the gene encoding the circumsporozoite protein (CSP) of Plasmodium falciparum from different transmission areas—XVI. Asembo Bay Cohort Project. Mol. Biochem. Parasitol. 125, 83–90 (2002).

    CAS  PubMed  Google Scholar 

  7. 7.

    Bailey, J. A. et al. Use of massively parallel pyrosequencing to evaluate the diversity of and selection on Plasmodium falciparum CSP T-cell epitopes in Lilongwe, Malawi. J. Infect. Dis. 206, 580–587 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Zavala, F., Cochrane, A. H., Nardin, E. H., Nussenzweig, R. S. & Nussenzweig, V. Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes. J. Exp. Med. 157, 1947–1957 (1983).

    CAS  PubMed  Google Scholar 

  9. 9.

    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  PubMed  Google Scholar 

  10. 10.

    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  PubMed  Google Scholar 

  11. 11.

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

    CAS  PubMed  Google Scholar 

  12. 12.

    Cohen, J., Nussenzweig, V., Nussenzweig, R., Vekemans, J. & Leach, A. From the circumsporozoite protein to the RTS, S/AS candidate vaccine. Hum. Vaccin. 6, 90–96 (2010).

    CAS  PubMed  Google Scholar 

  13. 13.

    The RTS,S Clinical Trials Partnership et al. A phase 3 trial of RTS,S/AS01 malaria vaccine in African infants. N. Engl. J. Med. 367, 2284–2295 (2012).

    Google Scholar 

  14. 14.

    The RTS,S Clinical Trials Partnership et al. First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N. Engl. J. Med. 365, 1863–1875 (2011).

    Google Scholar 

  15. 15.

    The RTS,S Clinical Trials Partnership et al. 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).

    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  PubMed  PubMed Central  Google Scholar 

  17. 17.

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

    CAS  PubMed  Google Scholar 

  18. 18.

    Oyen, D. 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).

    CAS  PubMed  Google Scholar 

  19. 19.

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

    Google Scholar 

  20. 20.

    Murugan, R. et al. Clonal selection drives protective memory B cell responses in controlled human malaria infection. Sci. Immunol. 3, eaap8029 (2018).

    PubMed  Google Scholar 

  21. 21.

    Scally, S. W. et al. Rare PfCSP C-terminal antibodies induced by live sporozoite vaccination are ineffective against malaria infection. J. Exp. Med. 215, 63–75 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Tan, J. et al. A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. Nat. Med. 24, 401–407 (2018).

    CAS  PubMed  PubMed Central  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  PubMed  PubMed Central  Google Scholar 

  24. 24.

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

    PubMed  Google Scholar 

  25. 25.

    Imkeller, K. et al. Antihomotypic affinity maturation improves human B cell responses against a repetitive epitope. Science 360, 1358–1362 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Dyson, H. J., Satterthwait, A. C., Lerner, R. A. & Wright, P. E. 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).

    CAS  PubMed  Google Scholar 

  27. 27.

    Ghasparian, A., Moehle, K., Linden, A. & Robinson, J. A. Crystal structure of an NPNA-repeat motif from the circumsporozoite protein of the malaria parasite Plasmodium falciparum. Chem. Commun. 3, 174–176 (2006).

    Google Scholar 

  28. 28.

    Batista, F. D. & Neuberger, M. S. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8, 751–759 (1998).

    CAS  PubMed  Google Scholar 

  29. 29.

    Foote, J. & Eisen, H. N. Breaking the affinity ceiling for antibodies and T cell receptors. Proc. Natl Acad. Sci. USA 97, 10679–10681 (2000).

    CAS  PubMed  Google Scholar 

  30. 30.

    Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).

    CAS  PubMed  Google Scholar 

  31. 31.

    Oyen, D. et al. Cryo-EM structure of P. falciparum circumsporozoite protein with a vaccine-elicited antibody is stabilized by somatically mutated inter-Fab contacts. Sci. Adv. 4, eaau8529 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Julien, J.-P. & Wardemann, H. Antibodies against Plasmodium falciparum malaria at the molecular level. Nat. Rev. Immunol. 19, 761–775 (2019).

  33. 33.

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

    CAS  PubMed  Google Scholar 

  34. 34.

    Flores-Garcia, Y. et al. Antibody-mediated protection against Plasmodium sporozoites begins at the dermal inoculation site. MBio 9, e02194-18 (2018).

  35. 35.

    Hayley, A. et al. Antibody feedback limits the expansion of cognate memory B cells but drives the diversification of vaccine-induced antibody responses. Preprint at https://doi.org/10.1101/808543 (2019).

  36. 36.

    Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001).

    CAS  PubMed  Google Scholar 

  37. 37.

    Sattabongkot, J. et al. Establishment of a human hepatocyte line that supports in vitro development of the exo-erythrocytic stages of the malaria parasites Plasmodium falciparum and P. vivax. Am. J. Trop. Med. Hyg. 74, 708–715 (2006).

    CAS  PubMed  Google Scholar 

  38. 38.

    Harris, C. et al. Polymorphisms in Anopheles gambiae immune genes associated with natural resistance to Plasmodium falciparum. PLoS Pathog. 6, e1001112 (2010).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Pompon, J. & Levashina, E. A. A new role of the mosquito complement-like cascade in male fertility in Anopheles gambiae. PLoS Biol. 13, e1002255 (2015).

    PubMed  PubMed Central  Google Scholar 

  40. 40.

    Tiller, T. 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).

  41. 41.

    Tiller, T. et al. Corrigendum to’Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning’. J. Immunol. Methods 334, 142 (2008).

    CAS  Google Scholar 

  42. 42.

    Wardemann, H. et al. Predominant autoantibody production by early human B cell precursors. Science 301, 1374–1377 (2003).

    CAS  PubMed  Google Scholar 

  43. 43.

    Ereño-Orbea, J. et al. Structural basis of enhanced crystallizability induced by a molecular chaperone for antibody antigen-binding fragments. J. Mol. Biol. 430, 322–336 (2018).

    PubMed  Google Scholar 

  44. 44.

    Kabsch, W. XDS. Acta Crystallogr. D. Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

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

    CAS  Google Scholar 

  46. 46.

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

  47. 47.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Morin, A. et al. Collaboration gets the most out of software. Elife 2, e01456 (2013).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Friesen, J. et al. Natural immunization against malaria: causal prophylaxis with antibiotics. Sci. Transl. Med. 2, 40ra49-40ra49 (2010).

    Google Scholar 

  50. 50.

    Gildenhard, M. et al. Mosquito microevolution drives Plasmodium falciparum dynamics. Nat. Microbiol. 4, 941–947 (2019).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank C. Canetta, J. Gaertner, A. Knauf, C. Winter and D. Foster (German Cancer Research Center, Heidelberg), C. Kreschel, L. Spohr, D. Eyermann and M. Andres (Max Planck Institute for Infection Biology, Berlin) and the DKFZ/European Molecular Biology Laboratory (EMBL)/Heidelberg University Chemical Biology Core Facility, especially P. Sehr, for technical assistance and services. The following reagents were obtained from BEI Resources, NIAID, NIH: HC-04, Hepatocyte (human), MRA-975, contributed by J. Sattabongkot Prachumsri. The work was supported by a Hospital for Sick Children Lap-Chee Tsui Postdoctoral Fellowship (S.W.S), a Canadian Institutes of Health Research (CIHR) fellowship (S.W.S), a CIHR Canada Graduate Scholarship – Master’s Award (E.T.), the Canada Research Chairs program (J.-P.J.) and the Bill and Melinda Gates Foundation (OPP1179906; J.-P.J, H.W. and E.A.L.).

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R.M., S.W.S., G.C. and E.T. designed and conducted the experiments, interpreted experimental results and wrote the paper. G.M. performed analyses and interpreted results. T.D., A.B. and K.P. conducted experiments. E.A.L., J.-P.J. and H.W conceived the study, designed and supervised the experiments, interpreted all results and wrote the paper.

Corresponding authors

Correspondence to Elena A. Levashina or Jean-Philippe Julien or Hedda Wardemann.

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A.B., K.P. and G.M. declare no conflicts of interest. R.M., T.D. H.W., G.C., E.A.L., S.W.S., E.T. and J.-P.J. have filed a patent application related to mAb 4493 and mAb 2541.

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Peer review information Alison Farrell was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Reactivity of human anti-PfCSP antibodies with different PfCSP peptides.

a, ELISA-reactivity of monoclonal human antibodies20 to KQPA, NPDP, NVDP, NANP and C-CSP at the indicated antibody concentrations. Red and green lines indicate positive (2A10; ref. 19) and negative controls (mGO5342), respectively. n indicates the number of tested antibodies. b, Binding strength of anti-PfCSP antibodies (n = 200; Supplementary Table 1) to the indicated overlapping peptides and C-CSP as in (a) is shown as calculated area under the curve (AUC) values based on ELISA measurements at different antibody concentrations. The frequency of reactive and non-reactive antibodies is indicated. c, t-SNE clustering-based illustration of the ELISA binding strength to the indicated peptides as determined by AUCs for all PfCSP-reactive antibodies (n = 200). d, Antibody affinities to the indicated PfCSP peptides and C-CSP measured by SPR. e, t-SNE clustering-based illustration of the affinity of PfCSP-reactive antibodies to the indicated peptides and C-CSP measured by SPR for antibodies with ELISA AUC values >5. n indicates the number of antibodies.

Extended Data Fig. 2 Ig gene usage of anti-PfCSP antibodies with different reactivity profiles and cross-reactivity of IGHV3-33-encoded antibodies.

a, IGHV and paired IGKV or IGLV gene usage of antibodies with the indicated binding profiles: NANP-specific, NANP, NVDP cross-reactive and NANP, NVDP, NPDP cross-reactive antibodies; C-CSP cross-reactive antibodies, and NVDP-specific and NVDP, NPDP cross-reactive antibodies. Antibodies using the same IGHV are indicated as solid lines around the pie charts. n in the pie chart centers indicate the number of antibodies. b, ELISA PfCSP-reactivity of monoclonal plasmablast antibodies20 (n = 111) tested at 4 µg/ml (left). The PfCSP reactivity of IGHV3-33-, IGKV1-5-encoded antibodies including mAb 2541 (n = 6) was confirmed at different concentrations and is indicated as mean area under the curve (AUC; center). Red and green filled circles indicate the positive (2A10; ref. 19) and negative (mGO5342) control mAbs, respectively. The binding profiles of the six PfCSP-reactive IGHV3-33-, IGKV1-5-encoded plasmablast antibodies to the indicated peptides and C-CSP are shown (right). Mean AUC values from three independent experiments are shown (center, right).

Extended Data Fig. 3 Comparison of IGHV3-33-encoded antibodies binding to NANP and C-CSP peptide with NANA motif.

a, Isothermal titration calorimetry (ITC) measurements of IGHV3-33-encoded antibodies binding to NANP3 (mAbs 2243, 2541, 3945, 4498) or a 14-aa long C-CSP peptide (PNRNVDENANANSA; mAb 3246). b, Detailed interactions between mAbs 2243 and 2541 and NANP5, mAbs 4498 and 3945 and NANP3, and mAb 3246 and NANA. H-bonds are shown as black dashes. c, Thermodynamic parameters for Fabs 1210, 2243, 2541, 3945, 4498 and 3246 binding to PfCSP peptides as determined by ITC. Experiments were performed in duplicate or triplicate. Standard error of the mean (s.e.m.) is reported. NB denotes no binding.

Extended Data Fig. 4 Delineation of PfCSP binding by mAb 4493 and comparison to other mAbs of reported structures.

a, ITC measurements of mAb 4493 with the indicated peptides. b, Thermodynamic parameters of Fab 4493 binding to PfCSP peptides. Experiments were performed in duplicate. s.e.m. is reported. c, Superposition of NPDP peptides recognized by Fab fragments of mAbs 4493 (green) and CIS4323) show that the peptides are recognized in a similar U-shaped conformation, but by different angles of approach. d-f, Overlay of peptide conformations observed in co-complexes with the indicated antibodies, for which structures were reported with multiple peptides. Information about the positioning of these motifs in the antibody paratope is provided. Three distinct paratope positions (-1, 1 and 2) are indicated. Amino acid residues resolved in the structures are underlined. d, CIS4323. e, CIS4223. f, 31118.

Extended Data Fig. 5 Comparison of the in vivo protective capacity of mAbs 4493, 317, and CIS43.

a, Capacity of mAbs 31718, CIS4323, 4493 and 121025 to protect mice from blood-stage parasitemia after passive i.p. mAb transfer (300 µg/mouse) and exposure to the bites of mosquitoes infected with Pb-PfCSP parasites. The percentage of parasite-free mice is indicated. The C-CSP-reactive non-inhibitory mAb 1710 (ref. 21) was used as negative control. Pooled data from two independent experiments is shown (N=2). The total number of mice per group is indicated (n). Statistical analyses were performed using Mantel-Cox log-rank test. Groups labeled with the same letter were not statistically significantly different. Groups labeled with different letters were statistically significantly different. b, Serum concentration of the transferred monoclonal antibodies in individual mice at the time of parasite challenge. Data shows the mean of at least two independent measurements. Red bars indicate mean values.

Extended Data Fig. 6 Cross-reactivity of mAbs 2A10, 580 and 663 determined by SPR.

Affinities of the chimeric mAb 2A10 with mouse variable and human IgG1 constant region19 and of mAbs 663 and 58019 to the indicated PfCSP peptides measured by SPR.

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Murugan, R., Scally, S.W., Costa, G. et al. Evolution of protective human antibodies against Plasmodium falciparum circumsporozoite protein repeat motifs. Nat Med 26, 1135–1145 (2020). https://doi.org/10.1038/s41591-020-0881-9

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