Structural studies of Plasmodium PIR proteins and their interactions with human inhibitory immune receptors

Malaria is one of the most deadly diseases to affect mankind, leading to around half a million deaths and hundreds of millions of cases each year. It is caused by infection with tiny Plasmodium parasites. These single celled organisms are injected into affected individuals through the bite of an infected mosquito and develop and divide within the human liver and blood. The symptoms of the disease occur during the blood stage of infection. Here the parasites invade human blood cells and divide within them, with one parasite entering each blood cell and ten to twenty emerging two days later.

An intracellular life style provides these parasites with major advantages. Pathogens within the blood are under constant attack from the human immune system and concealment within a host cell allows them to avoid detection. However, Plasmodium send a small number of parasite molecules to the surfaces of infected red blood cells. This is a dangerous strategy, as it risks their detection, and so these proteins have critical functions in helping the parasites to survive within the infected human. To help avoid detection, these surface molecules are not present in single copies, but as diverse protein families. This allows the parasite to respond when the human immune system learns to recognise one of their surface molecules by switching to use a different, unrecognised molecule.

The most commonly found proteins on the surfaces of malaria-infected blood cells are the PIR proteins. All types of Plasmodium parasites studied so far produce PIRs, including the major human-infective malaria parasites, Plasmodium falciparum and Plasmodium vivax. Although we know remarkably little about the roles that these proteins play during infection, recent studies have given new and tantalizing clues. In one such study, malaria infection was characterised in mice. Mouse malaria can either be acute, leading to severe illness, or chronic, with long term, low level disease. Whether the malaria episode became chronic or acute correlated with which of the PIR proteins was expressed. In parallel, two different groups of Plasmodium falciparum PIR proteins (also known as RIFINs) were found to bind to human molecules know as inhibitory immune receptors. These receptors dampen the human immune response by reducing the effects of immune cells, potentially reducing the capacity of these cells to recognise infectious agents. Indeed, binding of RIFINs to the human inhibitory immune receptor, LILRB1, reduced antibody production, which will make the immune system less responsive. These findings suggest the exciting hypothesis that the PIR proteins dampen the human immune system, reducing its ability to detect and destroy the parasite. This would aid parasite survival and transmission between individuals, causing more malaria cases.

Remarkably, no-one knows what a PIR protein looks like or how they bind to inhibitory immune receptors. This makes it extremely challenging to understand how they function and how they affect the human immune system. Without this insight it is hard to classify the hundreds of PIR proteins into groups or to work out which human receptor each group binds. Finally, without knowing their structures, we cannot understand which bits of the PIRs are similar and which are variable. If we wish to train the immune system to recognise all PIR proteins, then we need to be able to find their invariant parts. This funding will therefore allow us to address these questions, understanding the structures, functions and variability of the PIRs. This will help us to understand the role of the most commonly found protein on the surface of the malaria-infected blood cells, showing us how they modulate the immune system and revealing whether we can find an invariant site on these molecules which we can target therapeutically to as part of our quest to destroy this deadly parasite.

This project will use the expertise of the Higgins group at structural and functional characterisation of host-parasite interactions. This will be complemented by collaborators who bring expertise in genomic analysis, analysis of immune cell function and animal models of malaria infection.

PIR proteins, human receptors and antibody Fab fragments will expressed in a secreted form from mammalian cell culture systems. Monoclonal antibodies may be generated by immunizing mice, fusing splenocytes, selecting clonal hybridomas in semi-solid media and screening reactivity by ELISA. Fab fragments will be produced by papain cleavage. Proteins, antibodies and protein complexes will be purified by standard chromatography methods. Protein-protein interactions will be assessed by surface plasmon resonance, microscale thermophoresis and isothermal titration calorimetry. Structures will be determined by x-ray crystallography using molecular replacement, sulphur SAD and heavy metal-based approaches for phasing. Binding surfaces will be determined using hydrogen-deuterium exchange mass spectrometry and confirmed using mutagenesis. Sequence analysis will be conducted using MEME analysis with sequence determinants identified from structural features, or from sequences identified as containing ligand binding determinants.

This research will have a rapid and direct academic impact, in the UK and globally. In the long term, it may have an economical and societal effect in all regions of the world where malaria is prevalent, as it involves understanding the major protein family found on all malaria-infected blood cells. We expect impact within the academic community, through translation to guide vaccine development, through understanding inhibitory immune receptors, through training of postdoctoral workers, through research placements for school and undergraduates and through outreach activities.

Potential Translational Implications:
While this proposal does not have directly translational goals, its findings may have potentially significant implications for future therapeutic development. The PIRs are the predominant surface antigens found on Plasmodium infected erythrocytes and are exposed to the immune system throughout the blood stage of infection. This raises to the question of whether they will be targetable by therapeutics. To answer this, we need to know if they are too variable, or whether they have identifiable conserved features. The identification of a family of LAIR-1 containing antibodies which target a broad set of RIFINs gives hope to such an approach. Our approach, in which we combine structural studies with analysis of sequence diversity across the PIR families, will reveal whether or not these family members have conserved surfaces which can be targeted by future vaccination approaches. If we identify such a surface, we will use Rosetta-based protein design approaches to develop novel immunogens which elicit antibodies against these surfaces and will test their broadly inhibitory potential.

We are also aware that the inhibitory immune receptors to which the PIR proteins bind are important therapeutic candidates, as possible targets to dampen the immune system. Understanding how the RIFINs modulate these receptors has the potential to guide the development of future therapeutics which modulate human immune responsiveness.

While therapeutic development is unlikely to be part of the three years of this particular proposal, we will be vigilant in looking for opportunities to exploit our findings. In particular, we have access to assistance by Oxford University Innovations, an organization that exists to help Oxford-based academics to find partners to translate their research.

Training:
The research associate employed through this funding will receive training in structural biology, protein design and parasite biology. These skills are highly transferable to other medically relevant biological studies, either in academia or industry. They will also receive training in transferable skills, including communication, project management, writing and IT. This will equip them to make major contributions to science and technology in the future. They will be mentored in career progression throughout the three years and beyond to help them to exploit this training.

Education:
The applicant provides research opportunities for school and undergraduate research placements in their laboratories. These involve engagement in an original research project, allowing the students to develop laboratory experience. In many cases, this has resulted in students deciding to study biochemistry or to take a graduate research degree, equipping them to contribute to the scientific research base of the UK.

Outreach:
As described in the communications plan, the applicants arrange events to reach different groups of people who are not actively engaged in research. These individuals are fascinated to hear about the interactions of pathogens with their human hosts and the work proposed here will directly feed into our outreach presentations. In many cases these presentations are to school groups and provide opportunities to encourage these students to study medically relevant subjects at University.