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Unravelling the immune signature of natural malaria transmission blocking immunity by protein microarray

Final Report Summary - SIGNAL (Unravelling the immune signature of natural malaria transmission blocking immunity by protein microarray)

The transmission of P. falciparum from man to mosquito depends on the presence of mature malaria transmission stages (gametocytes) in the human peripheral blood. If these gametocytes are mature and present in a viable sex-ratio of male and female gametocytes, further development of malaria parasites in mosquitoes can render mosquitoes infectious upon their next bite. Natural and vaccine-induced immune responses can reduce the transmission of malaria from man to mosquitoes by interfering with gametocyte fertilization. The development of the limited number of malaria transmission blocking vaccine (MBTV) candidates has so far been limited by difficulties achieving correctly folded immunogenic proteins for vaccination. Two leading candidates, the pre-fertilization antigens Pfs48/45 and Pfs230, are expressed on the surface of mature gametocytes and elicit transmission reducing activity (TRA) in a fraction of individuals naturally exposed to malaria infection. It has been shown repeatedly that full TRA may be present in absence of immune responses to these well-characterised proteins, yet no attempts have been made to comprehensively identify the immune signatures of naturally acquired TRA. The SIGNAL project aims to unravel the immune signature of TRA. The specific objectives are to 1. determine the transmission blocking potential of serum samples of individuals naturally exposed to malaria in high throughput membrane feeding assays; 2. perform immune profiling of serum samples with and without evidence for naturally acquired TRA using a sexual stage specific 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.