Characterization of the CSS protein as a vaccine target in Plasmodium sporozoites

• Grant number: 24/17121-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2024
• End date: October 31, 2026
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Daniel Youssef Bargieri
• Grantee: Xiomara Alexandra Gaitán
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/06769-0 - Discovery of vaccine targets and compounds against Malaria transmission, AP.JP2

Abstract

Invasion of host cells by Plasmodium zoites is a complex process and involves multiple proteins and protein complexes that are targets for the development of malaria vaccines. In P. falciparum, a complex (PCRCR) composed of P. falciparum thrombospondin-related apical merozoite protein (PTRAMP), cysteine-rich small secreted protein (PfCSS), PfRh5-interacting protein (PfRipr), cysteine-rich protective antigen (CyRPA) and reticulocyte binding protein homologue 5 (PfRh5), is essential for merozoite invasion of erythrocytes. In P. knowlesi and other non-laverania Plasmodium, CyRPA and Rh5 are not conserved, and a trimeric complex (PCR) composed of PkPTRAMP, PkCSS and PkRipr was identified as essential for merozoite invasion. Pull-down of PbRipr from P. berghei merozoite extracts identified PbPTRAMP and PbCSS, showing that the complex is also present in P. berghei merozoites.Recently, we used the P. berghei mouse malaria model to study the role of PbCSS in invasive stages and as a potential vaccine antigen.Unsuccessful PbCSS knockout attempts indicated it is essential for merozoite invasion and suggest that the PCR is indeed a core invasion complex present in Plasmodium merozoites. However, vaccination of mice with PbCSS-based recombinant proteins or mRNA did not protect mice against challenge with P. berghei blood stages. Likewise, antibodies raised against the P. vivax CSS showed only modest inhibition of P. vivax invasion of reticulocytes in vitro.In P. berghei sporozoites, PbPTRAMP and PbCSS are expressed but PbRipr is absent, suggesting that the PCR complex is not formed and raising the question whether sporozoites use these proteins for invasion. We confirmed CSS expression in P. beghei and P. vivax sporozoites. To study the function of CSS in pre-erythrocytic stages, we are using the diCre rapamycin-dependent recombinase system to conditionally knockout (cKO) PbCSS in sporozoites. Preliminary experiments showed that while PbCSS-cKO sporozoites display normal mosquito salivary gland invasion and gliding, they are deficient in hepatocyte invasion and mouse infection. Mice vaccinated with PbCSS-based mRNA and challenged with sporozoites were partially protected, as evidenced by a one-day delay in patent parasitemia.Our data suggest that PbCSS may be a common component of different invasion complexes in merozoites and sporozoites, and is a promising target for the development of vaccines that disrupt multiple stages of the parasite life cycle.The workplan of the postdoctoral fellow will involve the generation of vaccines based on the PbCSS and PvCSS using different strategies (lentivirus, mRNA, adenovirus) to vaccinate mice. The work will also involve further investigation of the role of CSS in sporozoites through invasion experiments, as well as cell traversal. Using molecular tags, the goal is also to localize CSS in sporozoites and likely in sexual stages, since in silico data suggest the presence of CSS in gametocytes.Expected outcomes: we expect to describe a new important protein for sporozoite invasion into hepatocytes and target for vaccines, that may also be a target for transmission blocking antibodies.