In silico profiling of the plasmodium knowlesi 32 kDa antigen: Diversity, epitope prediction, and structural modeling.

The emergence of Plasmodium knowlesi malaria in Southeast Asia, particularly Malaysia, necessitates effective interventions. However, high polymorphism often limits the utility of key merozoite surface proteins (MSPs) as viable vaccine candidates. Pk32, a predicted glycosylphosphatidylinositol (GPI)-anchored protein in P. knowlesi, shares homology with the relatively conserved Plasmodium vivax Pv32, suggesting it may be a promising vaccine candidate. We analyzed the genetic diversity, polymorphism, and signatures of natural selection and 3-D structure using 39 full-length Pk32 sequences, primarily from Malaysian Borneo. Sequence analysis showed low nucleotide diversity (π = 0.0061) and limited polymorphism. Phylogenetic analysis indicated no geographical clustering. Natural selection tests; codon-based Z-test, tajima's D, codon-based site-by-site analysis (FEL, MEME, FUBAR, SLAC) provided strong evidence of purifying selection acting on Pk32, suggesting functional constraint alongside population expansion. In silico B-cell epitope prediction identified three common epitomes. Crucially, two epitopes (145PKERES150 and 167DIGKKQNS174) were conserved in all sequences. Mapping these conserved epitopes onto the predicted and refined 3D structure of Pk32 highlights specific stable targets. The observed combination of limited genetic variation and the presence of stable, conserved B-cell epitopes strongly suggests that Pk32 is a compelling candidate for inclusion in a blood-stage vaccine against P. knowlesi.