Structural basis for conserved and distinct antigen recognition by a lineage of malaria-protective antibodies

Monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) have demonstrated substantial promise in preventing malaria infection and disease. PfCSP is characterized by a central region composed of repetitive NANP motifs, which serve as major targets for protective antibodies. Several potent mAbs targeting this region exhibit homotypic Fab-Fab interactions, which enhance antigen binding and contribute to their neutralization potency. Among these, mAb 399, encoded by the IGHV3-49/IGKV2D-29 (VH3-49/VK2D-29) germline lineages, forms head-to-head inter-Fab contacts mediated primarily by germline-encoded residues. In this study, we determined high-resolution X-ray crystal and cryo-EM structures of two additional Fabs, derived from the same germline lineages, 7160 and 7118, in complex with PfCSP-derived peptides. Both Fabs bound the NANP6 repeats with high affinity (KD 5-10 nM). Fab 7160 formed similar inter-Fab homotypic interactions to Fab 399 upon binding to an extended repeat peptide (NANP), indicating a conserved mode of recognition. In contrast, Fab 7118, which features a longer CDRH3, does not form homotypic contacts. Instead, Fab 7118 induced a bend in the peptide and adopted a distinct binding mode, which prevents inter-Fab interactions. A cryo-EM structure of Fab 7118 in complex with a recombinant shortened PfCSP construct (rsCSP) further revealed potential disruptions at inter-Fab contact sites. These findings highlight the structural versatility of VH3-49/VK2D-29-derived antibodies and demonstrate that CDR loop variations within a shared antibody lineage can modulate antibody conformation, homotypic Fab-Fab interactions, and epitope engagement. Our study provides mechanistic insights into the diverse strategies by which CSP-specific antibodies can achieve high-avidity binding and protective immunity.