3D11 Fab production and purification: The resulting pcDNA3.4-3D11 Fab KC and −3D11 Fab HC or −3D11 Fab 58/73 HC plasmids were co-transfected into FreeStyle 293 F cells for transient expression using FectoPRO DNA Transfection Reagent, cultured in GIBCO FreeStyle 293 Expression Medium, and purified via KappaSelect affinity chromatography (GE Healthcare), cation exchange chromatography (MonoS, GE Healthcare), and size-exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare). Recombinant PbCSP production and purification: The resulting pcDNA3.4-PbCSP-6xHis, -PbC-CSP-6xHis and -PbCSP-αTSR-6xHis plasmids were transiently transfected in FreeStyle 293 F cells using FectoPRO DNA Transfection Reagent, cultured in GIBCO FreeStyle 293 Expression Medium, and purified by HisTrap FF affinity chromatography (GE Healthcare) and size-exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare).

Plasmodium sporozoites express circumsporozoite protein (CSP) on their surface, an essential protein that contains central repeating motifs. Antibodies targeting this region can neutralize infection, and the partial efficacy of RTS,S/AS01 - the leading malaria vaccine against P. falciparum (Pf) - has been associated with the humoral response against the repeats. Although structural details of antibody recognition of PfCSP have recently emerged, the molecular basis of antibody-mediated inhibition of other Plasmodium species via CSP binding remains unclear. Here, we analyze the structure and molecular interactions of potent monoclonal antibody (mAb) 3D11 binding to P. berghei CSP (PbCSP) using molecular dynamics simulations, X-ray crystallography, and cryoEM. We reveal that mAb 3D11 can accommodate all subtle variances of the PbCSP repeating motifs, and, upon binding, induces structural ordering of PbCSP through homotypic interactions. Together, our findings uncover common mechanisms of antibody evolution in mammals against the CSP repeats of Plasmodium sporozoites.