INVESTIGATING Pf-PHISTb AS TARGET FOR NATURALLY ACQUIRED IMMUNITY AND THEIR UTILITY IN BLOCKING GAMETOCYTE TRANSMISSION

Abstract

Introduction: Plasmodium falciparum is a major parasite causing malaria predominantly in sub-Saharan Africa region and a leading cause of morbidity and mortality. A lot of effort has been made to understand the malaria parasite in order to develop effective interventions against malaria. However, due to drug resistance and lack of highly efficacious vaccine, the problem persists calling for concerted efforts to manage the parasite. During the blood stage infection, the parasite exports hundreds of proteins to the infected red blood cell (iRBC) milleu. One group of such proteins is Plasmodium Helical Interspersed sub-Telomeric (PHIST) family whose role in malaria pathogenesis is yet to be fully understood although some of its members have been associated with vital parasite processes during the erythrocytic cycle. The PHIST family has three subfamily namely PHISTa, PHISTb and PHISTc. The aim of this study was to evaluate the immunological capacity of PHISTb proteins (PF3D7_0532400, PF3D7_1401600, and PF3D7_1102500) in malaria transmission and their potential as candidates for malaria transmission blocking vaccine. Methods: PHISTb gene PF3D7_1401600 was cloned and expressed as recombinant proteins using TA cloning strategy. Two other recombinant PHISTb proteins PF3D7_0532400 and PF3D7_1102500 were harvested after expanding their glycerol stock. Anti-malarial immunity to PHISTb proteins was evaluated by indirect ELISA using sera from infected individuals in a high malaria transmission region (Kisumu) in Kenya and a low malaria transmission region (Median) in Sudan. Associations between anti-PHISTb antibody responses and anti-Plasmodium falciparum-schizonts extract, a marker for malaria transmission, were evaluated. Polyclonal antibodies to PHISTb protein PF3D7_1401600 were generated in rats. The ability of the anti-PHISTb polyclonal antibodies to disrupt gametocyte growth development was evaluated in-vitro. vi Antibody levels between two independent groups were assessed using unpaired Wilcoxon test. Comparison between more than two groups was assessed using Kruskal-Wallis test and Dunn test for pairwise comparison. P < 0.05 was considered significant. Data analysis was conducted in R 4.1.1. Results: The expression of the PHISTb proteins, PF3D7_0532400 (50KDa), PF3D7_1401600 (45.5KDa), and PF3D7_1102500 (65KDa) was confirmed using western blot assay. Naturally acquired antibodies in sera from infected individuals from Kenya and Sudan reacted to the three expressed PHISTb proteins. This reactivity did not vary with malaria transmission intensity and it was not statistically significant (P > 0.05). There was a positive correlation between the anti-PHISTb of the three recombinant PHISTb proteins to the anti-Pf-schizonts of strains (NF54 and Dd2). The anti-PHISTb (PF3D7_1401600) polyclonal antibodies generated in rats showed the capability of disrupting gametocyte development in 1:100 and 1:50 dilutions in-vitro. Conclusion: The study showed that PHISTb are targets of natural acquired immunity and that anti-PHISTb production varies with malaria transmission intensity. Additionally, the study elucidated a positive correlation between anti-schizonts and anti-PHISTb suggesting a positive relationship between malaria exposure and levels of anti-PHISTb antibodies. Further, this study showed that natural immunity to PHISTb can disrupt gametocytes development although molecular confirmation is needed. In conclusion, P. falciparum PHISTb holds a great potential for the development of malaria transmission blocking vaccine. Keywords: PHISTb, Gametocytes, schizonts, Plasmodium falciparum