Insights into the biological constraints on malaria red blood cell invasion and the implications for vaccine development

Abstract

Malaria is a disease caused by Plasmodium species parasites transmitted between humans by a mosquito vector. Asexual parasite replication occurs inside red blood cells (RBCs) and is the cause of all clinical symptoms of malaria. Despite significant reduction in global malaria incidence since the year 2000, there was >200 million cases and >600,000 deaths in the year 2021. Development of an efficacious vaccine would provide another much-needed level of defence against this disease. One form of the parasite which could be targeted for vaccine development is the merozoite, the form of the parasite that invades the RBC. Merozoites are exposed to the host immune response prior to invading the next RBC, and malaria infected people naturally acquire antibodies to the proteins on the merozoite surface. Merozoite surface proteins (MSPs) have long been considered as potential vaccine candidates but are mostly still uncharacterised. They are likely involved in host RBC recognition/invasion, and they may provide protective epitopes for immunity development. In this thesis, I employed Cas9 gene editing to study both function and immunogenicity of MSP4 and MSP5 of P. falciparum, the most prominent human pathogen, and P. knowlesi. P. knowlesi is a zoonotic species which is in vitro culture adapted, and as such is used here as a surrogate to study another important human pathogen, P. vivax, which is yet to be culture adapted. MSP4 and MSP5 were targeted for genetic deletion, and in the instances where the gene was refractory to deletion, they were put under a conditional knock-out system. In the absence of PfMSP4 and PkMSP5, I assessed parasite blood stage replication which revealed some of the first direct evidence that MSPs have a role in RBC invasion. It also demonstrated that P. falciparum and P. knowlesi have opposite importance for MSP4 and MSP5. I generated chimeric P. knowlesi which functionally expresses the P. vivax alleles of MSP4 and MSP5, these lines were subsequently used to demonstrate that malaria exposed people generated antibodies specific to these proteins and that they may play a role in protective immunity. I also characterised P. falciparum MSP2 knock-out parasites, particularly how they have increased susceptibility to growth inhibition by antibodies targeting another, unrelated surface protein called apical membrane antigen 1 (AMA1). These studies also investigated the mechanism by which this sensitisation occurs, with studies suggesting that antibody size, but less so access to the target antigen, are factors that influence increased antibody potency with MSP2 knock-out. Gene editing was also employed to investigate the MSP7 paralogous gene family, a member of has been shown in P. falciparum to form the most abundant complex (with MSP1 and MSP3) on the merozoite surface. I attempted to delete P. knowlesi MSP7s, as well as express P. vivax MSP7 genes which are strong candidates for vaccine development. The approaches used for manipulation of the PkMSP7 locus were ultimately unsuccessful, however, they did demonstrate that P. knowlesi may require more than one functional paralog for survival. They have also highlighted the importance of considering parasite protein – protein interactions when generating chimeric parasite lines. The studies outlined in this thesis have shed new light on the function of several MSPs, their importance to merozoite invasion of the host RBC and developed several new geneedited parasite lines that can be used in prioritisation and development of merozoite vaccine candidates.