Final Report Summary - SIGNAL (Unravelling the immune signature of natural malaria transmission blocking immunity by protein microarray)
During the project, a molecular assay was developed to better quantify male and female gametocytes in the peripheral blood. This assay was successfully deployed in field samples from Burkina Faso, Mali, Cameroon and Kenya with a sensitivity below 0.1 gametocyte per microliter of blood and forms a valuable tool to understand the epidemiology and biology of malaria transmission stages and the impact of interventions on malaria sex ratio. In addition, we expanded our work on understanding naturally acquired TRA by screening the largest number of plasma samples to date for functional TRA. In order to be able to perform this large-scale screening, the gold standard assay to assess TRA, the standard membrane-feeding assay, has been optimised to allow high throughput phenotyping of plasma samples from malaria endemic regions. This was achieved using a parasite line that expresses green-fluorescent protein (GFP) and firefly luciferase (LUC). The GFP-LUC assay was used to determine the transmission blocking phenotype of 649 plasma samples from Cameroon, The Gambia and Burkina Faso. A fraction of these samples (n=22; 3.4%) possessed strong transmission-blocking properties. First, de confirmed the role of antibodies against Pfs48/45 and Pfs230 in natural TRA by purifying antibodies against these proteins and testing naturally acquired purified antibodies against Pfs48/45 and Pfs230 in the SMFA. This work provided the first direct evidence that responses against these proteins are causally related to TRA. Subsequently, we performed immune-profile for other antigens differentially recognized by these transmission-blockers and non-blockers. This was performed using a custom-made protein microarray. To this end we compiled a list of 319 P. falciparum proteins whose expression/transcription is up-regulated in mature gametocytes and which possess characteristics of surface-located/exposed proteins. These proteins were expressed in an E. coli based in vitro transcription-translation system and printed onto a nitrocellulose microarray. Probing the sera on the gametocyte array identified 42 proteins that individuals with TRA reacted to more intensely, and more commonly. Our analysis substantially increased the number of Plasmodium antigens implicated in the development of immune responses that inhibit of parasite development in mosquitoes, and provided multiple novel targets for future MBTV development. Immunization with the newly identified antigens is currently ongoing as part of a follow-on SIGNAL-II project. Taken together, we have developed a novel highly sensitive molecular assay for assessing gametocyte sex ratio in natural infections. Our TRA findings indicate that we can plausibly increase the number of target antigens for MTBV development and can determine the immune profile of naturally acquired TRA, which would be a great asset for epidemiological and clinical malaria studies.