Genetic Polymorphism of Plasmodium falciparum Circumsporozoite Protein in Kigali, Rwanda

Sandra Noukimi Fankem¹Mbonimpa Jean-Bosco²Edgar Mutebwa Kalimba^1,2Mariama Telly Diallo¹Jacob Souopgui^1,2*

• ¹Université libre de Bruxelles, Brussels, Belgium
• ²King Faisal Hospital, Kigali, Rwanda

Malaria remains a major public health challenge across sub-Saharan Africa, with Plasmodium falciparum responsible for the vast majority of cases and deaths. In Rwanda, although control measures have led to significant progress, malaria continues to be endemic, with urban centres like Kigali experiencing continuous transmission. With the recent rollout of malaria vaccines such as RTS,S and R21, understanding the genetic variability of vaccine-targeted antigens is essential for anticipating and enhancing vaccine performance. This study investigated the genetic diversity of the P. falciparum circumsporozoite protein (Pfcsp) gene among 245 clinical isolates collected between October 2021 and June 2023 at the referral King Faisal Hospital Rwanda in Kigali, using Oxford Nanopore Technology platform. A total of 48 distinct haplotypes were identified, indicating high haplotype diversity (Hd = 0.8899) but moderate nucleotide diversity (π = 0.00834), suggesting immune-driven balancing selection. The N-terminal region was highly conserved across isolates, including full conservation of the KLKQP motif, reinforcing its functional importance in hepatocyte invasion. In contrast, the central repeat region exhibited substantial variability in NANP/NVNP tetrapeptide repeat numbers, and the C-terminal region, particularly the Th2R and Th3R epitopes showed extensive polymorphism. Notably, fewer than 1% of sequences matched the 3D7 vaccine strain, and several key amino acid positions associated with vaccine escape showed high mutation frequencies. These findings suggest that the genetic divergence of circulating csp variants in Kigali could be one of several factors influencing vaccine performance, underscoring the importance of ongoing molecular surveillance to guide eventual vaccine implementation in Rwanda and other endemic regions.