CLONING AND EXPRESSION OF POTENTIAL VACCINE CANDIDATES FOR PLASMODIUM FALCIPARUM

Abstract

Introduction: Malaria remains a major global health burden, with Plasmodium falciparum responsible for the most severe and deadly cases. Despite current vaccine efforts, a fully protective and multistage vaccine remains elusive. This study focused on the cloning, expression, and evaluation of P. falciparum antigens as potential vaccine candidates targeting various life cycle stages of the parasite. Materials and Methods: Seven target genes encoding stage-specific antigens for circumsporozoite protein (CSP), thrombospondin-related ahesion protein (TRAP), apical membrane antigen (AMA-1), merozoite surface protein (MSP-1), falcipain (FAL), gametocyte surface protein (GSP) and transmission-blocking antigen (TBA) were synthetically constructed and codon-optimized for optimal expression in Escherichia coli. Signal peptide regions were removed to enhance expression efficiency and solubility. Four of the proteins namely AMA-1, MSP-1, FAL, and TBA were successfully expressed and purified. Cytotoxicity of the recombinant proteins was assessed using BJ human fibroblast cells treated with concentrations ranging from 1 to 200 μg/mL. Immunogenicity was evaluated in CD1 male mice assigned to groups of three, with each group receiving a mixture of adjuvant and a single or combination of the recombinant antigens, intramuscularly. The control group received an equivalent volume of adjuvant diluted in phosphate-buffered saline (PBS). The immunogenicity was then evaluated by indirect ELISA to measure antigen-specific antibody responses. Results: All four recombinant proteins exhibited successful expression and purification. ELISA results revealed that all proteins induced high antigen-specific antibody responses in immunized mice. Most importantly, the combination of antigens elicited higher antibody titers compared to individual antigen administration, suggesting a potential synergistic effect. Cytotoxicity assays showed low toxicity for the tested proteins, with BJ cell viability remaining above 70% even at the highest concentration tested, indicating biocompatibility. Conclusion: Malaria continues to pose a significant public health challenge in sub-Saharan Africa, where it accounts for most global cases and deaths, especially among children and pregnant women. The endorsement of two license malaria vaccines, RTS, S/AS01 and R21 represents a major milestone, however, both vaccines face limitations with modest efficacy. This study demonstrates the successful cloning, expression, and initial evaluation of four P. falciparum vaccine candidate proteins. Their low cytotoxicity and high immunogenic potential support their further development as components of a multistage malaria subunit vaccine. The promising findings of this study provide a foundational step toward developing a more effective, multistage malaria vaccine, which could play a critical role in reducing the disease burden and supporting elimination efforts in the most affected regions.