Genetic signatures of  Plasmodium vivax Circumsporozoite Surface Protein during malaria resurgence in Thailand

The Plasmodium circumsporozoite surface protein (CSP) is the best characterized pre-erythrocytic vaccine target for malaria. It is a multifunctional protein important for sporozoite mobility, mosquito salivary gland invasion, and hepatocyte invasion. We analyzed diversity of Plasmodium vivax CSP gene (pvcsp) during the 2022-2023 malaria resurgence in northwestern Thailand and assessed how pvcsp haplotypes may affect parasite development in the mosquitoes. Amplicon sequencing of 69 P. vivax isolates revealed both canonical pvcsp variants: VK210 (n = 66) and VK247 (n = 3). The VK210 type exhibited high polymorphism within the central repeat region, with 21 haplotypes (H1-H21) composed of 13-20 repeat motifs. Haplotype H2 was the most common, accounting for half of all VK210 sequences, and in membrane feeding assays with Anopheles dirus, appeared to produce more salivary-gland sporozoites per oocyst than other haplotypes, suggesting that repeat-region variation may modulate vector competence. Together, these findings report contemporary pvcsp diversity in Thailand's highest transmission area, provide functional evidence that repeat-region polymorphisms shape vector-parasite interactions, and highlight three globally prevalent motifs (GDRADGQPA, GDRAAGQPA, ANGAGNQPG) as prime targets for future PvCSP vaccines.