Mapping HLA-restricted T-cell epitopes within conserved regions of Plasmodium vivax circumsporozoite protein from Thailand

Plasmodium vivax is a major cause of global malaria morbidity, posing unique elimination challenges due to dormant liver-stage hypnozoites that cause clinical relapses. The development of an effective vaccine is a prerequisite for eradication, yet no licensed vaccine currently exists due to the parasite's complex life cycle, genetic diversity, immune evasion, and the lack of robust continuous in vitro culture. This study focuses on the circumsporozoite protein (PvCSP), a promising pre-erythrocytic target, to computationally identify HLA-restricted conserved T-cell epitopes and their corresponding population coverage. The blood spot samples (n=34) were collected from P. vivax -infected patients in Southern Thailand. The PvCSP gene was amplified using nested PCR and sequenced via Illumina NGS to assess genetic polymorphism. Deduced amino acid sequences of the Thai clinical isolates and 281 publicly available global reference sequences from Brazil and Vanuatu were aligned against the P. vivax Salvador I reference strain to assess genetic conservation across functional domains. CD4 +‬ and CD8 +‬ T cell epitopes and population coverage were predicted using the Immune Epitope Database (IEDB) against prevalent Thai HLA class I and class II alleles to prioritise predicted strong HLA-binding peptide candidates.‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ Computational mapping of the conserved region of PvCSP identified multiple CD4 + ‬ and CD8 + T cell epitopes predominantly concentrated within the conserved N- and C-terminal regions, whereas the central repeat region (CRR) contained only limited predicted CD4 + T cell epitopes. Further sequence logo analysis of nine conserved amino acid residues in predicted T cell epitopes revealed that both HLA class I (HLA-A, -B, -C) and class II (HLA-DRB1, -DQA1/-DQB1)-restricted epitopes were universally conserved across diverse geographical isolates, suggesting their potential as candidates for further evaluation in the development of broadly protective vaccines. Population coverage analysis, based on regional HLA allele frequencies, demonstrated that these cumulative PvCSP-derived T cell epitopes confer high population coverage across diverse populations, achieving 95.86% in Southeast Asia.‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ Based on the positional distribution of predicted binding peptides, the conserved N- and C-terminal regions of PvCSP contained a high density of both CD4 + ‬ and CD8 +‬ T cell epitopes, whereas the genetically diverse CRR harbored only a limited number of predicted CD4 + T cell epitopes. Mapping these epitopes against prevalent regional HLA alleles demonstrates extensive population coverage across Southeast Asia. These findings provide a foundation for a multi-epitope vaccine to limit parasite immune evasion and support P. vivax malaria elimination.‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬