A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein

Abstract

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.

Figure 1. Immunization with PfSPZ Vaccine induces robust antibody responses in malaria-exposed individuals

a, Protocol of PfSPZ immunization of Tanzanian adults. b, Binding of serum IgM and IgG antibodies to PfSPZ. Median fluorescence intensity (MFI) values are for binding at a 1/1000 serum dilution (representative of n=2 independent experiments). Samples in red, black and blue are from protected (U, G, V, H), non-protected (NP) and placebo (C) volunteers, respectively. Results for donor W are not shown as this donor was immunized with a lower dose of PfSPZ. c, Staining of PfSPZ by serum from a European blood donor, serum from a protected individual (donor H) and a monoclonal antibody (MGU8) (representative of n=3 independent experiments). d, Dose-dependent binding of three representative antibodies to PfSPZ measured by flow cytometry (representative of n=2 independent experiments). e, Binding values of the panel of IgG monoclonal antibodies to PfSPZ (representative of n=2 independent experiments). The values indicate the concentration of antibody required to reach a 10,000 MFI. f, Number of PfSPZ-binding IgG and IgM monoclonal antibodies isolated from protected donors G and U. g, Number of mutations in the heavy chains of IgG (n=19 antibodies) and IgM (n=65 antibodies) isolated from the Tanzanian volunteers. These values were calculated by adding the number of VH and JH mutations. Results are shown as mean ± s.d.. A two-sided t-test was used to compare the number of mutations.

Figure 2. Highly neutralizing antibodies use VH3-30^f genes and exhibit dual specificity for NANP and the N-terminus junction

a, In vitro inhibition of PfSPZ traversal and invasion by monoclonal antibodies (n=2 or 3 independent experiments). Bars show mean percentages relative to control experiments with irrelevant human IgG. b, Correlation of invasion with binding affinity to PfSPZ (from n=1 representative experiment out of 2). A two-tailed Spearman’s correlation was performed, 95% confidence interval 0.2878–0.8887. c, In vivo activity of monoclonal antibodies in humanized liver mice infected by P. falciparum via mosquito bites (n = 4 or 5 mice per antibody tested). Bars show the mean percentage of liver parasite burden relative to that of control mice injected with irrelevant human IgG. Error bars show s.d.. A one-sided ANOVA with Kruskal-Wallis test was used; *P≤0.05, **P≤0.01, ****P<0.0001. d, Scheme of PfCSP (not to scale). e, Binding of monoclonal antibodies to full-length PfCSP and PfCSP peptides (representative of n=2 independent experiments). The antibodies are classified according to the VH gene used and the residue at position 52 for VH3-30^f antibodies. The antibodies in the box are those tested in the in vivo assay. Antibodies belonging to the same clone are highlighted in the same colour. NANP18, NANPNANPNANPNANPNA; NPDP15, KQPADGNPDPNANPN; NPDP19, KQPADGNPDPNANPNVDPN. 22-110 and 282-383 are long N-terminal and C-terminal peptides from Pf3D7 CSP, respectively^,. f, Correlation of affinity for NPDP15 with in vivo antibody efficacy. A two-tailed Spearman’s correlation was performed (from n=1 representative experiment out of 2). Confidence interval not determined by Prism for n<10.

Figure 3. Somatic mutations increase affinity for PfCSP and lead to the acquisition of dual specificity

a, VH and VL genealogy trees of MGU1 and MGU10. Shown are the nucleotide and amino acid substitutions, with the latter in parentheses. UCA, unmutated common ancestor; BP, branch point. b–e, Binding of members of the MGU1 and MGU10 clonal family to PfCSP, PfSPZ, NANP18, and NPDP15, respectively (representative of n=2 independent experiments). f, Binding of MGU10 mutants to NANP18, NPDP15 and full-length PfCSP (representative of n=2 independent experiments). g, Binding of monoclonal antibodies to NPDP19 peptide mutants (n=1 experiment). The letters highlighted in red show the mutated residues in the peptide.

Figure 4. Structural basis for recognition of an N-terminal junctional peptide by MGG4

a, Binding interface of MGG4 in complex with the ¹KQPADGNPDPNANP¹⁴ peptide; only residues 7 to 13 of the peptide have interpretable electron density (indicated in bold). The peptide is shown in the cartoon representation with sidechains as sticks, while the heavy and light chains of MGG4 are shown as dark and light grey surfaces respectively. The CDR loops are in the cartoon representation: CDRH1 (green), CDRH2 (blue), CDRH3 (magenta), CDRL1 (light green), CDRL2 (light blue) and CDRL3 (pink). The W52 sidechain is shown as blue sticks and the interfacial waters are highlighted as red spheres. b, Buried surface area (BSA) for the heavy chain (HC) and light chain (LC) with the peptide. c, BSA for individual peptide residues with the Fab. d, Pseudo 3₁₀ turn for the DPN motif of the bound peptide (yellow carbons) and type I β-turn for the previously published crystal structure of the unbound ANPNA peptide (green carbons). Stabilizing hydrogen bonds between the sidechain of D9/N2 and the amide backbone of N11/N4 in the two structures are highlighted by the dashed line. e, 2Fo-Fc electron density map for the N-terminal peptide contoured at 2.0σ (dark blue) and 0.8σ (light blue).