Date of Award

January 2025

Document Type

Open Access Thesis

Degree Name

Master of Public Health (MPH)

Department

School of Public Health

First Advisor

Amy K. Bei

Second Advisor

Sunil Parikh

Abstract

Malaria remains a major global health burden, with an estimated 263 million cases and597,000 deaths reported in 83 countries in 2023 (1). While P. falciparum is responsible for the most severe cases, non-falciparum species such as P. vivax, P. ovale curtisi, P. ovale wallikeri, P. malariae, and P. knowlesi also pose significant public health risks, particularly in co-endemic regions. Current malaria vaccine efforts mainly focus on P. falciparum which is reasonable because over 90% of the global malaria-related morbidity and mortality are caused by P. falciparum (2). However, laying emphasis on targeting P. falciparum leaves a critical gap in cross- species protection, because the non-falciparum species are also prevalent in many endemic regions and contribute significantly to the global malaria burden through mechanisms such as relapse, chronic infection, and asymptomatic infection (3). Therefore, it is important to develop strategies that address both P. falciparum and non-falciparum species if we want to achieve comprehensive and sustainable malaria elimination. Identifying conserved antigens with potential for broad- spectrum malaria immunity is essential for advancing malaria vaccine development.

Malaria pathogenesis is closely tied to the blood-stage of the parasite’s life cycle, wheremerozoites invade human erythrocytes, multiply, and cause the clinical manifestations of disease. This process of erythrocyte invasion is not only central to symptom development and disease severity but also represents a key target for vaccine and drug interventions. A major advance in our understanding of this invasion mechanism came with the identification of the PCRCR complex. This complex is a conserved five-protein assembly composed of PTRAMP, CSS, Ripr, CyRPA, and RH5 which facilitates merozoite entry into red blood cells in Plasmodium falciparum by binding the receptor Basigin (4). Notably, while RH5 is unique to P. falciparum, another component of the complex, called Ripr, is conserved across multiple Plasmodium species (4, 5). In P. knowlesi, which lacks RH5, Ripr instead interacts with distinct invasion ligands, highlighting the functional plasticity and evolutionary significance of this complex (4). These findings indicate the importance of Ripr as a central, conserved component of the invasion machinery, making it a promising candidate for cross-species malaria vaccine development (Figure 2 and 3).

PfRipr is a key component of the PCRCR complex in P. falciparum, where it plays anessential role in stabilizing the interactions between the merozoite and the host erythrocyte during invasion (4). Given its conservation across multiple Plasmodium species, PfRipr has emerged as a promising candidate for cross-species malaria vaccine development (5). However, functional evaluation of PfRipr orthologs from non-falciparum species remains challenging, as most of these parasites cannot be continuously cultured in vitro (6). To overcome this limitation, alternative model systems such as P. knowlesi, which is genetically tractable and capable of in vitro culture, offer a valuable platform to assess the functional conservation and vaccine potential of PfRipr across species.

This study focuses on generating a transgenic chimeric P. knowlesi model to assess thepotential of Ripr protein as a cross-species vaccine candidate. Since most non-falciparum Plasmodium species cannot be continuously cultured in vitro, assays relying on isolated parasites are limited in scope, short-lived, and often highly variable. In contrast, a transgenic approach using P. knowlesi enables stable genetic replacement of endogenous genes with orthologs from other species in a tractable, human-compatible culture system. This provides a controlled and reproducible platform for functional analysis of conserved antigens such as Ripr and allows for deeper investigation of their role in erythrocyte invasion and potential as universal vaccine targets. The research follows a structured approach, beginning with in silico design of CRISPR guides for Ripr chimeras using Geneious. This is followed by cloning experiments using designed guides, transformation into E. coli, and subsequent plasmid extraction. Plasmids are then sent for sequencing, and alignment verification is performed to identify successfully edited constructs. Donor templates, incorporating 500 bp upstream and downstream homologous regions, are designed for recombination and submitted for synthesis to facilitate further transgenic modifications in P. knowlesi (Figure 1). While this project does not include antibody testing or invasion assays, it establishes a foundational transgenic platform for future functional studies on the Ripr protein. These transgenic lines can be used in downstream assays to assess cross-species functional conservation and immune recognition. For example, monoclonal antibodies described in studies such as Healer et al. and the recent Seager preprint could be employed to evaluate the inhibitory effects on invasion, providing critical insight into the potential of Ripr as a broadly protective vaccine target (5, 7). The successful generation of transgenic P. knowlesi expressing PfRipr orthologs will provide an essential tool for evaluating its role in erythrocyte invasion and its potential as a vaccine target in future experiments. This research contributes to advancing the application of genetic tools for malaria vaccine development and lays the groundwork for future investigations into cross-species malaria immunity.

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This is an Open Access Thesis.

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