A comprehensive survey of protein-protein interactions between Plasmodium falciparum merozoites and human receptors

Malaria is one of the most common infectious diseases in the world, with more than 300 million cases of malaria each year, leading to more than 1 million deaths, primarily in children under the age of five in Africa. Malaria is caused by single-celled Plasmodium parasites and one species is particularly deadly - Plasmodium falciparum - which is responsible for almost all malaria deaths. Plasmodium parasites have a complex life cycle, but all the symptoms of malaria are caused when they invade human red blood cells, also known as erythrocytes. The parasite uses the erythrocyte as a source of food, multiplies inside it, and, after 48 hours, breaks open the erythrocyte to release multiple new parasites. This cycle of invasion, multiplication and re-invasion results in high numbers of parasites in the blood stream.

Because Plasmodium parasites spend the majority of their life cycle inside human cells, they are difficult to target with vaccines, and there is currently no licensed vaccine for malaria. However, the process of erythrocyte invasion is one of the few stages of the parasite's life cycle when they are exposed to the host immune system, and is therefore a potential target for vaccine-induced invasion-blocking antibodies or drug development. For vaccines or drugs to be developed, it is critical that we understand which proteins on the parasite surface are binding directly to erythrocytes and the identity of the erythrocyte proteins that they are binding to. By first identifying these parasite-erythrocyte interactions, it may be possible to develop novel therapeutics that prevent invasion and hence cure or prevent malaria. Research over the last two decades has led to the identification of numerous P. falciparum proteins that may be involved in erythrocyte invasion, but only in very cases do we know which erythrocyte proteins they bind to. This is in large part because of the technical challenges involved: protein-protein interactions between the surface of cells are often very short-lived (lasting only a few seconds) and so are very difficult to detect using standard experimental approaches.

At the Wellcome Trust Sanger Institute we have recently developed a new approach to detect short-lived protein-protein interactions. Called AVEXIS (Avidity based Extracellular Interaction Screen), it uses libraries of proteins, expressed and purified in the lab, to detect novel interactions. Over the last two years we have started to apply this to the process of P. falciparum erythrocyte invasion and have created small pilot libraries of erythrocyte and P. falciparum proteins to screen for interactions. This preliminary work has identified two new parasite-erythrocyte interactions, one of which appears to play a very important role in erythrocyte invasion. However, this initial screen was limited in scope and we still do not have binding partners for the majority of P. falciparum proteins. In this research we will expand our libraries of parasite and erythrocyte proteins, and also include other components of the host blood which P. falciparum parasites may be interacting with, such as sugar structures that are present on cell surfaces. After screening the parasite and blood component libraries for interactions in an all vs. all manner, we will test whether any newly discovered interactions have roles in erythrocyte invasion using P. falciparum parasites that can be cultured in the lab. This research will build up the first systematic map of interactions between P. falciparum proteins and human blood cell components, and will identify protein-protein interactions that are of particular interest for vaccine or drug development. We will also deposit the DNA constructs that are used to create our recombinant protein libraries in not-for-profit reagent resource collections, where they can be freely accessed by other researchers and thereby empower malaria research in many labs across the globe.

Plasmodium falciparum parasites cause more than a million deaths from malaria each year. All the symptoms and pathology of malaria are caused by the blood stages of P. falciparum development, which are initiated when merozoites invade human erythrocytes. Erythrocyte invasion is potentially an attractive vaccine target because it is essential to parasite survival and the extracellular ligands that catalyse it are exposed on the parasite surface. Although many candidate P. falciparum ligands have been identified, in very few cases are their host receptors known, in part because cell surface protein-protein interactions are often of very low affinity, making them difficult to detect using standard approaches.

We will address this important gap in our understanding using AVEXIS, a protein-protein interaction technology developed at the Sanger Institute specifically to detect low affinity interactions. A pilot screen has already identified two novel P. falciparum-erythrocyte interactions, including one that plays a critical role in invasion. To expand these studies, we will:
1) Construct a comprehensive library of recombinant P. falciparum merozoite proteins
2) Screen the library against human erythrocytes to identify erythrocyte binding proteins
3) Identify carbohydrate receptors for merozoite ligands by screening glycan arrays
4) Identify protein receptors by using AVEXIS to screen libraries of human erythrocyte receptors or other soluble blood components
5) Validate the function of identified interactions using in vitro cultured P. falciparum parasites.
This approach will generate the first biochemically validated map of interactions between P. falciparum merozoite proteins and human blood cell components, and will identify protein-protein interactions that are of particular interest for vaccine or drug development. All expression constructs will be deposited in not-for-profit reagent resource collections to empower research in other labs.

Malaria is responsible for around 1 million deaths annually and the emergence and spread of drug-resistant strains is now a major global health concern. The economic cost of this disease is significant, estimated to represent up to 1.3% of GDP in countries with high transmission rates. The overarching, long-term goal of this research is to make a contribution towards an effective and cost-effective preventative treatment for malaria. The development of an effective vaccine for this disease would have a huge impact on global health and is a top priority for many public and privately led funding initiatives.

Scientific researchers working in the immediate subject area will be clear beneficiaries of this work, as outlined in the 'Academic Beneficiaries' section. In the context of translational impact, this research will be of particular benefit to researchers working within the global malaria vaccine initiatives both in the public and private sector. Merozoite proteins are regarded as excellent vaccine candidates. Should a single or multi-component blood-stage vaccine be an effective treatment for malaria, it is highly likely that one or more of the proteins within our recombinant merozoite protein resource will be a component of such a vaccine. Given our success in high level expression of functional proteins, the experience of expressing antigens in this project will be of clear utility to other vaccine projects focusing on the same targets. Furthermore, we have already shown that the combination of biochemical and in vitro approaches can make important functional insights into the role of merozoite proteins. The results of this work will therefore also help to rationalise antigen targets on the basis of function, and thereby provide a rational focus for vaccine development.

The ultimate aim of this research will be to identify candidate antigens that would lead towards a highly effective blood-stage vaccine, whether driven by ourselves or others. When this is achieved, the primary beneficiaries of this research would be the many billions of people living in malaria-endemic regions. A successful vaccine against malaria would therefore have a huge global health impact. This proposal is aimed at making a contribution towards this goal by expressing and determining the function of P. falciparum merozoite proteins. The timescale for any identified target/s to be used in a licensed vaccine is likely to be 10 to 20 years. The economic impact of an effective anti-malarial vaccine would be enormous, relieving a huge healthcare, economic and social burden from some of the poorest countries in the world, and also enabling non-native people travelling to and working in these countries to be effectively protected.

More broadly, the approach of producing recombinant protein libraries representing the surface repertoire of differentiated cell types to identify the receptors for pathogens is one that could be applied to other stages of the Plasmodium lifecycle, which also present important intervention targets. One important example is transmission-blocking vaccines, which have been identified as a high priority as part of the move towards malaria elimination. The same approaches could also be applied to other pathogens of global public health importance, including other parasites as well as viruses and bacteria.

Researchers employed on this grant would gain important skills necessary for producing and elucidating the function of P. falciparum merozoite proteins, which could play an important role in their own career development. These would include the expression systems used to produce the proteins and also the skills to culture and perform functional assays on P. falciparum parasites. The wider dissemination of these skills may lead to important insights in the function of merozoite proteins in other research environments.