A chimeric circumsporozoite protein exhibits strong seroreactivity in plasmodium falciparum and plasmodium vivax infections

Malaria remains a major global health challenge, demanding new strategies for effective and broad-spectrum vaccine development. Circumsporozoite Protein (CSP) is the primary target of pre-erythrocytic malaria vaccines such as RTS, S/AS01 and R21/Matrix-M, though their species-specific design limits protection mainly to Plasmodium falciparum . In this study, an immunoinformatics-guided multi-epitope chimeric CSP construct was designed by integrating B-cell, CTL, and HTL peptide epitopes from all human malaria parasites (P. falciparum , P. vivax , P. ovale , P. malariae , and P. knowlesi). Computational analysis predicted the construct to be stable, soluble, antigenic, and non-allergenic. Molecular Dynamics simulations revealed stable interactions between the chimeric Circumsporozoite Protein (CSP) and the Toll Like Receptor 4 (TLR4). Immune simulations predicted strong humoral and cellular responses characterized by high IgG titers and IFN-γ/IL-2 secretion. The recombinant chimeric CSP was successfully expressed in E.coli , purified under denaturing conditions and successfully refolded as confirmed through CD spectroscopy revealing a predominantly α-helical structure. Enzyme Linked Immunosorbent Assay (ELISA) with sera from P. falciparum and P. vivax infected patients demonstrated significant cross-reactivity, with higher seroreactivity observed for P. vivax samples. Interestingly, a significantly higher seroreactivity with refolded chimeric CSP compared with denatured chimeric CSP against P. vivax sera samples were observed suggesting that conformational epitopes dominate antibody recognition in P. vivax , compared with P. falciparum . Additionally, the chimeric CSP showed a significantly higher seroreactivity compared with full length Pv CSP. Collectively, these findings highlighted the immunogenic potential of the chimeric CSP construct and suggests its possible application as a serological surveillance marker in malaria-endemic regions.