Introduction

Malaria is disease caused by parasites of the Plasmodium genus. Four major species are witnessed to cause disease in humans, P. falciparum, P. vivax, P. ovale and P. malariae. Of these four species, P. falciparum is the most dominant, especially in Africa. According to the World Health Organization (WHO), 32 sub-Saharan Africa countries account for ~93% of Malaria-related deaths globally in 2021, of which P. falciparum are responsible for >99% of cases (1). Further, 2021 was the first year in over 20 years to exhibit an increase in Malaria cases and deaths per capita, likely due to COVID-19 disruptions in Malaria prevention. Regardless of the current pandemic’s influence on Malaria trends, per capita cases and deaths have remained steady since 2015, indicating the need for additional interventions, such as vaccines.

P. falciparum has a rather complex pathology. Its infection is characterized by three stages: liver, blood, and mosquito. Within each stage, the parasite takes on multiple physiologies that utilize a diverse range of receptors, signaling pathways, hosts, etc. The current leading vaccine candidate, RTS,S, targets the circumsporozoite protein found on the liver-stage sporozoite (2,3). While RTS,S is the only WHO-recommended vaccine to date, it has several drawbacks. Most notably, RTS,S has waning efficacy per year and does not guarantee sterilizing immunity (3-5); if the parasite progresses past the liver stage and infects erythrocytes (i.e., enters the blood stage), it undergoes an asexual reproduction cycle that bursts erythrocytes and contributes to most symptoms of clinical Malaria.

To combat these drawback, research is invested in blood-stage vaccine candidates that could work solo or in tandem with RTS,S. P. falciparum has also evolved strategies to evade antibodies that target the pre-blood-stage merozoite, such as redundant invasion pathways, surface protein polymorphism, and low immunogenicity of crucial antigens. One surface protein, apical membrane antigen-1 (AMA1), is naturally immunogenic and does not involve redundant invasion pathways (7,8). Its function is still not clearly defined, but it is thought to help with the engulfment of merozoites by erythrocytes in what is termed the “moving junction” (Figure 2). Importantly, AMA1 has been deemed crucial to merozoite invasion, as limited AMA1 expression does not allow P. falciparum to invade erythrocytes (9).

Figure 2 – The proposed hypothesis of merozoite invasion of erythrocytes. Merozoite surface proteins (MSPs) are thought to initiate primary contacts with erythrocytes and signal the merozoite a target cell is detected. Reticulocyte binding protein homologues (RHs) and erythrocyte binding antigens (EBAs) are proposed to reorient the merozoite and face its apex towards the erythrocyte, alongside initiating the moving junction formation, which is a formation that allows the merozoite to be engulfed inside the erythrocyte. Finally, AMA1 is thought to facilitate the entry of merozoites into the erythrocyte, allowing the merozoite asexual reproduction stage to begin. Figure from (8).

Like all Malaria antigen candidates to date, AMA1 has major drawbacks. Specifically, AMA1 is known for extensive polymorphism, with up to 64 known amino acid residues with recorded polymorphism (10,11). Furthermore, AMA1 has allelic polymorphism, and literature has identified 200+ haplotypes across African specimens (12). Several papers have identified lead antibody candidates that are strain-specific, with polymorphisms at specific residues causing resistance against other strains (10,13,14). Most of these polymorphic residues are dimorphic, but others are very diverse, with 3-6 alternate amino acids (Figure 3). AMA1’s hypothesized binding site, a hydrophobic trough between domain I and II, is not polymorphic. However, several loops surrounding the trough on each sides contain many amino acids known to be polymorphic, further complicating vaccine efforts.

Figure 3 – Polymorphic resides on the surface of AMA1. Dimorphisms are shown in orange (low frequency) and yellow (high frequency). Highly polymorphic regions are shown in red and disordered regions are shown in violet. We see the non-polymorphic and polymorphic faces of AMA1 in (a) and (b), respectively. Figure from (11).

Two major vaccine candidates have been evaluated to date, of which have utilized AMA1 from P. falciparum 3D7 and FVO variants (13,15,16). Given the extensive polymorphism described, these vaccines did not significantly improve clinical malaria episodes in their clinical trials. One of these two vaccines, FMP2.1/AS01a, exhibited slight strain-specific efficacy in a trial of Mali children (16). This data, alongside the previously mentioned antibody neutralization studies, argue for a vaccine that elicits antibodies against multiple AMA1 variants. Many vaccine designs could potentially achieve this, such as (A) targeting conserved regions, (B), including multiple AMA1 variant antigens in a single vaccine, or (C) targeting a specific epitope that may induce broadly neutralizing antibodies.

Our project aims to characterize polymorphism across domains I, II, and III of P. falciparum AMA1 sequences submitted to UniProt in order to characterize both polymorphism and conservation data across this antigen. Our project utilizes multiple bioinformatics tools to evaluate these trends, including amino acid frequency determination, phylogenetic tree analysis, and protein structure prediction. Overall, we hope this project helps improve bioinformatics approaches surrounding Malaria vaccines, especially for highly polymorphic antigen candidates, and influence future vaccine engineering efforts.