Structure guided design of a transmission-blocking malaria vaccine targeting Pfs48/45

Malaria is one of the most devastating infectious diseases to affect humankind. It kills around half a million people, mostly young children in Africa. It also places a huge disease burden on large parts of the world, leading to hundreds of millions of serious infections. As well as directly causing suffering and death, this limits the development of large parts of the globe, reducing productivity and maintaining inequality.

Malaria is caused by a tiny, single celled parasite, known as Plasmodium. An individual contracts malaria when bitten by an infected mosquito. The parasites are injected into the blood stream as the mosquito feeds. These first develop and divide within the liver without causing disease. They next emerge from the liver and infect red blood cells, replicating and increasing in number and driving the symptoms of malaria. At the same time, a fraction of the parasites within the blood take a different developmental route, adopting a form known as the gametocytes. When a mosquito takes a blood meal from an infected person, they are likely to ingest some of these gametocytes. Within the midgut of the mosquito these develop into male and female gametes, and fuse together. This completes the infection cycle and the parasites move to the salivary glands of the mosquito, ready to be injected into another human victim.

Development of a vaccine to prevent malaria has proved very challenging and it is likely that the vaccines of the future will simultaneously target multiple stages of the parasite life cycle, blocking both liver and blood cell entry. One component of such a vaccine is likely to target the gametes of the parasite and to stop them from fusing. This is known as a transmission-blocking vaccine component as it will prevent the development of the parasite within the mosquito and will therefore stop the disease from being passed from person to person through the action of this blood-sucking insect.

We study a molecule called Pfs48/45 that is found on the surface of the gametocytes and gametes of Plasmodium parasites. Pfs48/45 is essential for a male gamete to fuse with a female gamete and if the immune system of an animal is exposed to Pfs48/45, it produces molecules called antibodies that bind to Pfs48/45 and prevent gametes from fusing. This means that if we can include Pfs48/45 in a vaccine, it will trigger the human body to make antibodies that will prevent malaria from being transmitted to other people. This will reduce the prevalence of malaria in the community and will help to eradicate the disease.

Despite this promise, Pfs48/45 is a challenging molecule to produce in a functional form and in large quantities. In addition, if we are to make vaccines that simultaneously contain multiple components, it will be important for each component to be as small and focused as possible, to make it easier and cheaper for them to be produced and distributed. For this reason we aim to understand the structure and shape of Pfs48/45 and also to understand the location and the nature of the sites where the gamete-fusion-preventing antibodies bind. This information will allow us to use the latest computational tools to design novel molecules which contain just the regions of Pfs48/45 that are needed to bind to inhibitory antibodies.

We will then inject mice with these novel immunogens and study the antibodies that are produced in a mosquito-infection experiment. If mosquitoes are fed on human blood containing malaria parasites, gametes can fuse, leading to the formation of cysts in the gut wall of the mosquito. If gamete fusion is prevented, the cysts are no longer formed. We will therefore study the ability of the antibodies induced by our novel immunogens to prevent cyst formation revealing which of our designs is most effective at blocking gamete fusion and preventing transmission of the malaria parasite. These newly designed molecules will form part of the malaria vaccines of the future.

This project combines the expertise of the Higgins and Biswas groups, allowing us to conduct a work plan which progresses from structural characterization of an important parasite surface protein through to the testing of designed immunogens in a pre-clinical setting.

We will produce Pfs48/45 in stable Drosophila s2 cells. Existing hybridomas will be used to make monoclonal antibodies. New monoclonal antibodies will be generated by immunizing mice, fusing splenocytes to make hybridomas, selecting clonal colonies in semi-solid media and screening for Pfs48/45 reactivity by ELISA. Fab fragments will be produced by papain cleavage and Pfs48/45:Fab complexes will be assembled and purified by size exclusion chromatography. The structure of Pfs48/45 will be determined by x-ray crystallography and key antibody epitopes will be mapped by crystallography and by hydrogen-deuterium exchange mass spectrometry.

Stabilised versions of Pfs48/45 will be generated using the PROSS-design algorithm. Novels immunogens will be made by grafting key epitopes from Pfs48/45 onto scaffold proteins and will be optimsed using Rosetta-based modeling. These immunogens will be produced in E. coli and used to immunize mice. The sera will be tested for the ability to prevent oocyte development in mosquitos using a membrane-feeding assay with parasite-infected blood.

Pfs230 domains will be generated in transiently transfected stable Drosophila s2 cells and will be tested for binding to Pfs48/45 by surface plasmon resonance. Complexes will enter into crystallization trials. In addition, existing Pfs48/45 antibodies will be used to immuo-purify Pfs48/45:Pfs230 complexes from cultured gametocytes. These will be studied by electron microscopy, initially using negative stain and a T12 microscope, and then by electron cryo-microscopy using a Talos Arctica microscope with a K2 detector. Imaging processing will be done using RELION to obtain three-dimensional reconstructions.

The research proposed here 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 the design of immunogens that can be included in vaccines to prevent the transmission of malaria parasites, in particular Plasmodium falciparum and Plasmodium vivax. We expect impact within the academic community, through translation to guide vaccine development, through training of postdoctoral workers, through research placements for school and undergraduates and through outreach activities.

The academic community:
This work will address the important questions of how Plasmodium species undergo gamete fusion and how antibodies can prevent this. Structural studies of Pfs48/45 and its complex with Pfs230 will be of interest to researchers studying the sexual stage of Plasmodium and to those studying the mechanisms of gamete fusion more broadly. A major part of the proposal also involves design of novel protein immunogens that can be used to raise protective immune responses. These studies will not only be of great interest to researchers involved directly in the development of transmission-blocking malaria vaccines, but will also be of significant value to those engaged in protein design and structure-guided vaccinology against many infectious agents. We will communicate findings throughout the project, through local seminars, visits to institutes, presentations at conferences and open access publications.

Translational work:
A major component of this proposal is directly translational as it involves pre-clinical vaccine design and testing. Our structural studies of Pfs48/45 form an important basic biosciences project. However, we aim to immediately progress this work towards structure-guided immungen design, using the latest protein design methods, to make novel immunogens which present the key inhibitory epitopes required to elicit transmission blocking antibody responses. We will then test these immunogens by raising antibody responses in immunized animals and testing their efficacy at preventing oocyst formation in infected mosquitos. This work will directly develop components that will form part of the multi-stage malaria vaccines of the future.

Training:
The postdoctoral research fellow 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 applicants provide 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 groups 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 are also opportunities to encourage these students to study medically relevant subjects at University.