A bivalent multi-epitope vaccine targeting CSP and TRAP of Plasmodium vivax designed through immunoinformatics and structural bioinformatics.

Malaria associated with Plasmodium vivax is still one of the main public health concerns due to relapse-associated infections. Moreover, the absence of a broadly effective licensed vaccine makes the situation more alarming. In this scenario, epitope-based vaccine design helps to improve safety, immunogenicity, and population coverage. The integrated workflow of immune informatics and structural bioinformatics was used to establish a bivalent multi-epitope vaccine that targets pre-erythrocytic antigens, circumsporozoite protein (CSP), and thrombospondin-related adhesive protein (TRAP). Antigenicity, allergenicity, toxicity, and transmembrane topology were evaluated before epitope prediction. B-cell, MHC class I, and MHC class II epitopes were identified, screened, and their antigenic origins were suggested. The vaccine construct was assembled by using linkers and an adjuvant. Population coverage analysis, physicochemical characterization, three-dimensional structure prediction, refinement, and validation help to strengthen the goal. Molecular docking with Toll-like receptors (TLR2 and TLR4), immune response simulation, codon optimization, and in silico cloning were performed. The final construct was predicted to be antigenic, non-allergenic, nontoxic, and structurally stable, with broad global population coverage (98.8%). Docking analyses predicted potentially stable interactions with both TLR2 and TLR4. Immune simulation suggested coordinated innate and adaptive immune activation. The feasibility of expressing a protein was predicted using codon optimization and cloning analysis studies. The current study suggests a rationally engineered multi-epitope bivalent vaccine candidate against P. vivax, providing a base for future experimental validation.