Evidence for a model of conformational change by the Plasmodium falciparum circumsporozoite protein during sporozoite development in the mosquito host through the use of camelid single-domain antibodies.

Plasmodium sporozoites (SPZs) are formed in the Anopheles mosquito midgut from where they travel to the salivary glands and subsequently to the mam malian liver after deposition into the skin.The SPZ's main surface antigen, the cir cumsporozoite protein (CSP), plays a pivotal role in SPZ biology and constitutes the immunodominant target for host antibodies.In this study, we raised single-domain antibodies (sdAbs) against CSP from P. falciparum (PfCSP) by immunizing two alpacas with recombinant versions of the antigen.We found that all identified sdAbs specifically target PfCSP's globular TSR domain without cross-reacting with P. berghei CSP.Further characterization revealed that most sdAbs recognize native PfCSP on the SPZ surface, although they do not have any inhibitory effect on hepatocyte binding and invasion.Structural studies showed that all binders target the previously identified -epitope, confirming the non-protective nature of this epitope.Comparison of sdAb binding to midgut and salivary gland SPZs revealed a shift in the exposure and accessibility of the -epitope.Hence, our findings provide further evidence that CSP undergoes structural changes during SPZ development in the mosquito host.KEYWORDS malaria, Plasmodium falciparum, sporozoite, circumsporozoite protein, camelid single-domain antibodies M alaria is a devastating disease that presents an immediate health threat for almost half the world's population, with an estimated 263 million cases and 597,000 deaths in 2023 (1).The endemic 'hot spot' is Sub-Saharan Africa, which accounts for 94 and 95% of all malaria cases and deaths, respectively.The disease is caused by parasites of the Plasmodium genus and vectored by female Anopheles mosquitoes.The parasite sexually reproduces in the mosquito midgut and generates thousands of sporozoites (SPZs), which travel to the salivary glands where they await injection into the skin of the mammalian host.From their inoculation site, the SPZs travel to the liver where they differentiate into tens of thousands of merozoites.The latter re-emerge in the circulation to replicate within erythrocytes and cause the notorious malaria pathology (2).Most human malaria cases concern infections by P. falciparum, which causes the deadliest malaria.Due to the fast emergence of resistance against antimalarial drugs and insecticides (3, 4), intensive research has been performed during the past decades in the quest to design an effective vaccine (5).Recently, the first malaria vaccines have been approved (Mosquirix, also known as RTS,S/AS01, and R21/Matrix-M), which are subunit vaccines based on the P. falciparum circumsporozoite protein (PfCSP) (6, 7).