mRNA vaccines expressing Plasmodium falciparum CSP with distinct antigen localisation profiles induce differential humoral immunity and partial protection in a mouse sporozoite challenge model

Pre-erythrocytic malaria vaccines aim to prevent infection at the earliest stage of parasite entry by blocking Plasmodium falciparum sporozoites before hepatocyte invasion, thereby halting progression to symptomatic blood-stage disease. The circumsporozoite protein (CSP), the dominant surface antigen of sporozoites, remains the leading target for pre-erythrocytic vaccine development. Here, we investigated the use of an mRNA vaccine platform to express CSP and examined how antigen localisation influences immunogenicity and protective efficacy. We compared mRNA constructs encoding soluble CSP with versions engineered for membrane anchoring using either a mammalian glycosylphosphatidylinositol (GPI) sequence derived from placental alkaline phosphatase or a heterologous transmembrane (TM) domain from influenza H1N1 haemagglutinin. Immunisation with CSP mRNA-LNP formulations showed that membrane association altered the magnitude and profile of antibody responses, with distinct effects observed across the NANP repeat and C-terminal regions of CSP. Following intravenous challenge with transgenic Plasmodium berghei sporozoites expressing P. falciparum CSP, both soluble and membrane-associated vaccines delayed parasitaemia and conferred partial protection, but membrane targeting did not consistently improve protective efficacy. These findings show that antigen localisation can shape the immune response to mRNA-expressed CSP, while also highlighting that enhanced immunogenicity does not necessarily translate into greater protection.