Cryo-EM mapping of circumsporozoite protein epitope accessibility and visualization of antibody-induced shedding on Plasmodium falciparum sporozoites
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The circumsporozoite protein (CSP) on the surface of the sporozoite stage of Plasmodium falciparum parasites is one of the major malaria antigens recognized by the adaptive immune system. Antibodies against CSP can provide protection from malaria infection following mosquito bite of the human host. Recent studies have shown how such antibodies recognize recombinant, truncated forms of Plasmodium falciparum CSP (PfCSP); however, much remains unknown about epitope accessibility and presentation of PfCSP on the parasite itself. Understanding the nature of the native antigen is of significant interest for optimization of immunogen design, since immunization with whole attenuated sporozoites has been shown to provide long-lasting protection against malaria disease. Here we use cryo-electron microscopy and tomography to investigate how antibodies against various PfCSP epitopes (N-terminal domain (NTD), Junction, central repeat, and C-terminal domain (CTD)) impact the PfCSP coat on whole Plasmodium falciparum sporozoites (PfSPZ). We observe that PfCSP by itself cannot be visualized on the surface of the cell by cryo-EM. However, by titrating in antibodies that recognize the Junction motif and central repeats, we delineate a boundary layer formed by PfCSP on the sporozoite outer membrane. The antibody concentration series also enabled us to determine conditions required to induce the dramatic CSP shedding reaction. By comparing matched monovalent FAb with IgG, we demonstrate that antibody bivalency is critical to driving this reaction. Additionally, we demonstrate that even at high concentrations, antibody against the NTD does not bind, indicating the absence or inaccessibility of this epitope on the PfCSP displayed by radiation-attenuated sporozoites, while the CTD can be bound by antibodies without producing a CSP shedding reaction. These studies shed light on the nature of the PfCSP coat in its native context on the parasite, thus revealing the sporozoite’s antigenic profile.
AUTHOR SUMMARY
Studies of anti-malaria antibodies targeting the circumsporozoite protein (CSP), the dominant antigen on the surface of sporozoites that are transmitted from mosquito to human hosts, have focused primarily on their interaction with recombinant CSP fragments. A number of such antibodies have been shown to provide protection in animal models and in early human clinical trials. However, little is known about how the antibodies directly interact with CSP on whole sporozoites (SPZ). Using cryo-electron microscopy and cryo-electron tomography we examined how antibodies against major CSP epitopes recognize native CSP on intact Plasmodium falciparum SPZ. Understanding the accessibility of CSP epitopes on SPZ can illuminate the nature of the native antigen and indicate which epitopes are most effective to target through vaccination in order to produce a specific functional response such as CSP shedding. Additionally, we investigated the effects of antibody concentration and bivalency on antibody-induced CSP crosslinking and shedding. These findings can guide future vaccine immunogen design and help identify mechanisms of action for CSP-targeting antibodies.
