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


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).


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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.).

Author information




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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Competing interests

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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