A structure-based epitope tagging approach identifies vulnerable sites on the malarial P36-P52 protein complex for...

Malaria is caused by apicomplexan parasites of the genus Plasmodium, which are transmitted through the bite of Anopheles mosquitoes that inject sporozoites (SPZs) into the skin. SPZs migrate to and infect the liver for an initial round of replication. SPZs and liver stages have long been considered as ideal targets for malaria vaccines. The main SPZ surface protein, the circumsporozoite protein (CSP), is the target of currently approved malaria vaccines and prophylactic antibody therapies. Studies in rodent malaria models have shown that anti-CSP antibodies exert their protective effect mainly in the skin, but some of the most potent anti-CSP monoclonal antibodies show additional protective effects in the vasculature and liver. Other SPZ proteins involved at different steps of the infection process may thus represent additional targets for antibody-mediated neutralization. Three 6-cysteine (6-Cys) domain proteins (P36, P52 and B9) play an essential role during SPZ invasion of hepatocytes, yet their molecular function and whether they can be targeted by neutralizing antibodies remains unknown. Here, to fill this gap, we combined an integrative structural biology approach with functional experiments in the P. berghei rodent malaria model. AlphaFold-based structural modeling and experimental data obtained through electron microscopy and small-angle X-ray scattering indicated that the P36-P52 heterodimer displays a head-to-tail architecture with an interaction interface that is largely conserved among Plasmodium species. The structural models supported the rational design of an epitope tagging approach, which, combined with neutralizing assays, revealed that antibodies against tagged P36 and P52 can efficiently block invasion of hepatocytes by SPZs in culture conditions. The data show that the inhibitory activity of anti-tag antibodies heavily depends on epitope tag position and revealed that antibody-exposed vulnerable sites lie on the membrane-distal side of the P36-P52 heterodimer. In contrast, antibodies targeting tagged B9 had no inhibitory effect on SPZ invasion, irrespective of epitope positioning. These data show that the invasion step could be targeted by antibodies and indicate that the P36-P52 complex might be considered as a potential target for the development of next generation pre-erythrocytic malaria vaccines or therapeutic antibodies.