Insilico identification, characterisation of plasmodium falciparum hypothetical proteins and their assessment as molecular targets for malaria vaccine

Abstract

This thesis focused on malaria vaccinology and employed reverse vaccinology and molecular techniques to predict potential vaccine candidates against P. falciparum malaria. Reverse vaccinology is fundamentally an in silico method that predicts vaccine candidates from an entire organism’s proteome, enabling the identification and characterization of vaccine candidates in a realistically shorter time when compared to conventional vaccinology approaches. The need to control P. falciparum malaria is urgent because over 241 million people were infected with the disease worldwide and about 627,000 died in 2020 and yet it is also clearly documented that there is no effective malaria vaccine on market. The only clinically recommended vaccine against P. falciparum infection called RTS, S vaccine (Trade mark, MosquirixTM), offers suboptimal (30-50%) protection to clinical infection which is reported to wane within six months. This implies that there is need to identify an efficacious malaria vaccine candidate. There are about 47% (2538/5300) of the P. falciparum proteins whose functions are not yet known (as known as hypothetical proteins) but these could be of biological importance in regard to vaccine development. Thus their characterization could reveal potential vaccine candidates for development of an effective malaria vaccine. In this study, sequences of 23 hypothetical proteins were downloaded from the NCBI and rigorously screened using bioinformatics techniques for characteristics of vaccine candidates including; surface location (WoLFPSORTv2.0), antigenicity (Vaxijen2.0), nonhomology to human proteome (BLASTp), presence of signal peptide (SignalPv5.0), less than one transmembrane domains (TMHMMv2.0 and HMMTOPv2.0) and non-allergenicity (AllerTopv2.0 and AllergenFPv1.0), non-toxicity (ToxinPred). Three, XP-001350955.1, XP-001351030.1 and XP-001351047.1 out of the 23 HPs were predicted as potential vaccine candidates and their secondary and tertiary structures determined using SOPMA & PSIPRED and SWISS-MODEL. The two most highly promiscuous epitopes, 6KIFLFFIVL and 12FIVLEILLL from XP-001350955.1 were identified. The hypothetical protein, XP-001351030.1 was successfully produced as a recombinant protein (recXP-001351030.1 of ~28 kDa) in E. coli and was immunogenic in rabbits. The anti-recXP-001351030.1 antibodies recognised the native P. falciparum protein by Western blot. The antibodies also significantly inhibited P. falciparum merozoite invasion of human erythrocytes in the in-vitro invasion inhibition assay (IIIA). This thesis identified three PVCs and experimentally characterized XP-001351030.1 hypothetical protein from P. falciparum 3D7 strain. Challenge experiments, toxicity and safety studies in laboratory animals are recommended to determine its vaccine potential which can be used to facilitate the rapid formulation of a novel malaria vaccine.