Plasmodium knowlesi Duffy binding protein alpha region II (PkDBPαII) in clinical isolates from Peninsular Malaysia and Malaysian Borneo exhibit different immune responses in animal models

Plasmodium knowlesi utilizes the Duffy binding protein alpha (PkDBPα) to facilitate its invasion into human erythrocytes. PkDBPα region II (PkDBPαII) from Peninsular Malaysia and Malaysian Borneo has been shown to occur as distinct haplotypes, and the predominant haplotypes from these geographical areas demonstrated differences in binding activity to human erythrocytes in erythrocyte binding assays. This study aimed to determine the effects of genetic polymorphisms in PkDBPαII to immune responses in animal models. The recombinant PkDBPαII (~ 45 kDa) of Peninsular Malaysia (PkDBPαII-H) and Malaysian Borneo (PkDBPαII-S) were expressed in a bacterial expression system, purified, and used in mice and rabbit immunization. The profile of cytokines IL-1ra, IL-2, IL-6, IL-10, TNF-α, and IFN-γ in immunized mice spleen was determined via ELISA. The titer and IgG subtype distribution of raised antibodies was characterized. Immunized rabbit sera were purified and used to perform an in vitro merozoite invasion inhibition assay. The PkDBPαII-immunized mice sera of both groups showed high antibody titer and a similar IgG subtype distribution pattern: IgG2b > IgG1 > IgG2a > IgG3. The PkDBPαII-H group was shown to have higher IL-1ra (P = 0.141) and IL-6 (P = 0.049) concentrations, with IL-6 levels significantly higher than that of the PkDBPαII-S group (P ≤ 0.05). Merozoite invasion inhibition assay using purified anti-PkDBPαII antibodies showed a significantly higher inhibition rate in the PkDBPαII-H group than the PkDBPαII-S group (P ≤ 0.05). Besides, anti-PkDBPαII-H antibodies were able to exhibit inhibition activity at a lower concentration than anti-PkDBPαII-S antibodies. PkDBPαII was shown to be immunogenic, and the PkDBPαII haplotype from Peninsular Malaysia exhibited higher responses in cytokines IL-1ra and IL-6, antibody IgM level, and merozoite invasion inhibition assay than the Malaysian Borneo haplotype. This suggests that polymorphisms in the PkDBPαII affect the level of immune responses in the host.