Utilizing gametocyte immunity to reduce malaria transmission

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

The continuing success of the current malaria elimination campaign requires novel tools to efficiently block human infection and subsequent transmission of the parasite to mosquitoes. Current transmission blocking strategies target the development of the parasite in the mosquito stage, requiring complicated mosquito feeding readouts to measure efficacy. We recently identified the bone marrow as the major site of transmission stage development during infection. Based on this finding we hypothesized that the underlying host parasite interactions could be exploited to block parasite transmission. Indeed our preliminary studies demonstrate natural immune responses against parasite surface antigens and their functionality in terms of immune clearance. Here we utilize this new understanding to systematically define the immunity targeting malaria transmission with the ultimate goal of prioritizing a set of novel transmission blocking vaccine candidates.

Technical Summary

Malaria transmission from man to mosquito requires the uptake of male and female gametocytes from an infected host during a mosquito blood meal. Gametocytes are therefore essential for successful onward infection to new hosts and a major target for interventions. Our recent work demonstrates that gametocytes, together with a subset of asexual parasites, develop in the hematopoietic environment of the bone marrow before being released into circulation as transmission competent mature forms. Our preliminary studies have revealed natural immune responses targeting immature gametocytes that are correlated with reduced gametocyte burden in malaria patients. Using a combination of surface proteomics and immune profiling of immunised mice we have identified a series of putative surface antigens on gametocyte-infected red blood cells. The majority of them is shared with asexual stage parasites suggesting shared function in host cell interactions during development in the bone marrow. Here we propose to systematically investigate the targets and functionality of natural gametocyte immunity as a basis for novel vaccine development. For this purpose, we will combine a series of field studies in malaria endemic areas and experimental human infections, in vitro functional assays to define antigen-specific antibody activity and B-cell epitopes, as well proof-of-principle experiments in the rodent malaria model.

Planned Impact

The recent gains in malaria control have contributed to a renewed interest in malaria elimination and worldwide eradication. Thirty-five countries have fewer than 1000 malaria cases per year and are actively pursuing malaria elimination. In most African settings, malaria elimination is considered unrealistic with the currently available tools such as firstline treatment with artemisinin combination therapy (ACT), deployment of insecticide treated nets and indoor residual spraying. Tools or approaches that specifically aim to reduce malaria transmission are considered highly valuable for elimination efforts in these settings. In addition, the genuine threat of ACT resistant malaria parasites in the Greater Mekong Subregion has highlighted the need for interventions that can reduce and prevent the spread of (resistant) malaria parasites.

Malaria transmission blocking vaccines are high on the priority list of international academic groups that set the research agenda for malaria eradication (e.g. malERA), policy makers including the World Health Organization and funders with an interest in malaria vaccination or elimination strategies (e.g. PATH Malaria Vaccine Initiative and the Bill & Melinda Gates Foundation). The current project answers fundamental questions about malaria biology and immunology, which in itself forms an excellent justification for academic research. In addition, it forms a solid basis for future vaccine development projects. If successful, the current project will open new avenues for malaria vaccine development and vaccination strategies. All current and historic efforts to develop malaria transmission blocking vaccines focus on gamete antigens or mosquito antigens. The current project is the first to directly target gametocytes prior to ingestion by mosquitoes. A vaccination approach that aims to prevent gametocyte formation or maturation has utility as stand-alone intervention in settings of resistant malaria, epidemic or seasonal malaria and may be of particular use when combined with other vaccines that target other malaria life stages. When combined with pre-erythrocytic or bloodstage malaria vaccines, a vaccine targeting (the formation of) gametocytes may protect the partner vaccine and augment its community impact in reducing the force of infection.

The investigators and collaborators have solid networks that involve the above mentioned stakeholders, maximizing the chance that the findings from the current project will result in follow-up funding, are disseminated to policy makers at the highest possible level (as well as at a lower level to national malaria control programmes and country managers) and are shared with academic think tanks on malaria elimination and eradication.

Publications

10 25 50
 
Title Batch screen of host RBCs for immune recognition assays 
Description Variation in host RBC has a big impact in serum recognition of P. falciparum infected RBCs (iRBC). Some RBC batches are nonspecifically bound by anti-human secondary antibodies not specific to the parasite. To minimise this interference and reduce background binding, batches of RBCs are regularly screened for cross-reactivity anti-human antibodies using flow cytoemtry. iRBCs are stained using known positive and negative controls as well as the anti-human secondary antibody. Batches that show high background are discarded and only batches wilt low nonspecific background binding are collected for bulk parasite preparation and cryopreservation for downstream assays. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? No  
Impact This method help to minimise batch to batch variation of samples preps and therefore less noise in the data. The parasite samples generated can then be used across sites within the grant and ensure more standardised data. Reducing variation across assays, sites and cohorts will enable easy data comparison. 
 
Title Gametocyte enriched protein microarray 
Description A bespoke protein microarray was designed at Radboud UMC and LSHTM to assess novel antibody responses to putative gametocyte antigens. Inclusion of proteins on the array was determined with a pipeline criteria related to gametocyte specificity, likelihood of membrane association/presentation, and prior empirical/annotated evidence. Un-supervised inclusion was based principally on a gametocyte enrichment score generated to enable the arrays design [PMID: 29323249]. In short, the specificity of any protein for the gametocyte stage was scored by determining how often it had been detected across 11 proteomic analyses. Proteins were binned from low to high abundance and weighted according to the retrieval rates of proteins in two curated lists of 'gold standard' gametocyte and asexual genes, consisting of genes that are known to be specific for either asexual stages (n = 45) or gametocytes (n = 41). High expression of gametocyte gold standard proteins with concurrent absence of non-gametocyte gold standard proteins resulted in a high gametocyte score, calculated from the fraction of retrieved gametocyte genes over retrieved non-gametocyte genes. All scores were log-transformed and summed over all data sets. Specific criteria for inclusion were: 1. Proteins with evidence for presence or enrichment in gametocytes (score >0) PLUS presence of transmembrane domains (tm), signal peptides (sp), or GO term indicating membrane or surface expression (excluding mitochondrial proteins) [n=372] 2. Proteins without unique peptides in any proteomic analysis PLUS high gametocyte transcript score (>9.63), PLUS presence of TM/SP/GO term indicating membrane or surface expression (excluding mitochondrial proteins) [n=36, cumulative total = 407] 3. Gametocyte protein score >-10 (some evidence of gametocyte expression) PLUS presence of GPI anchor [22] or predicated export protein (PlasmoDB) [22] [n=60, cum. total = 436] 4. Proteins putatively exported by and specific to early or late gametocytes [n=30, cum. total = 443] 5. Gametocyte protein score of >9.69 (Gametocyte enriched), regardless of protein structure/function, and an average peptide score of 9 in each of the gametocyte databases [n=107, cum. total = 518) 6. Gametocyte specific proteins from early literature PLUS seropositive in field sera in >50% of samples after transmission season or with >20% sero-prevalence increase after transmission season (n=43, cum. total 451) 7. Gold standard gametocyte proteins in list used to generate transcript and protein score [n=41, cum. total = 538) 8. A priori protein selection. Markers of sexual stage exposure, TBV candidates, 6-cys proteins, proteins implicated in gamete fertility, asexual markers etc. (n=37, cum. total = 561) 9. Inclusion based on previous gametocyte array analysis (SIGNAL project) of differently reactive proteins in naturally occurring transmission blockers [n=45, cum. total = 580), 10. Inclusion based on association with the giRBC surface of immature gametocytes (PMID: 31167926): KEY PILOT STUDY [n=47, cumulative total and total number of IVTT targets on array = 600]. The array has been printed in two iterations: by the university of California Irvine (UCI), in collaboration with Prof Phil Felgner, and by Antigen Discovery (Irvine, CA). 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact The array will be used exclusively by our research teams. Pilot studies assessing the immune response to gametocyte antigens in controlled human malaria infections indicate significant recognition of giRBC surface proteins. Array analyses will be performed on samples with and without giRBC surface recognition as identified in the gametocyte flow cytometry assays developed during this project and detailed elsewhere. 
 
Title Malawi cohort study 
Description 140 participants (6m and older) living in 4 clusters of 6-8 households enrolled in a cohort study beginning in November 2021. Participants are visited every two weeks to obtain finger prick blood samples including: blood smear to detect asexual and gametocyte stage parasites, dried blood spot on filter paper for molecular detection of parasites, whole blood in RNA preservative for to detect gametocyte specific mRNA and markers of sexual commitment, and plasma for anti-gametocyte antibody screening. If gametocytes are detected, venipuncture blood is obtained for membrane feeding assays. Twice during cohort follow-up, PBMCs will also be collected. 
Type Of Material Biological samples 
Year Produced 2021 
Provided To Others? No  
Impact Samples will be used the the assays described in the other outcomes. 
 
Title Mice sera immunized with early gametocyte proteins 
Description Mice were immunized with six early gametocyte proteins to evaluate immunogenicity and generate antibodies. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact Sera were obtained from mice immunized for six different early gametocyte proteins. These sera will be assessed to evaluate expression of the different proteins on infected RBCs and their immunological potential against early stage gametocytes. 
 
Title Plasmodium falciparum surface serum recognition assay using frozen parasite preparation 
Description We have developed a protocol to generate and cryopreserve parasite material for use in flow cytometry and live microscopy assays. Late stage asexual parasites and stage I-II gametocytes are magnetically purified to enrich for parasite infected RBCs (iRBC). iRBCs are cryopreserved using a Sorbitol-Glycerolyte solution (28% Glycerol, 3% Sorbitol, 0.65% NaCl in Distilled water). 1x106 iRBC cells/ml per vial are mixed with human serum and Sorbitol-Glycerolyte solution then frozen in liquid nitrogen until needed. To measure serum surface recognition by flow cytometry frozen parasite preparations are thawed from liquid nitrogen in a water bath at 37°C for 1-2 min. NaCl solutions of lowering concentration (12% NaCl and 1.6% NaCl) are added dropwise to the parasite preps. Parasites are then washed 3 times using incomplete media and visually assessed by giemsa stain. The samples are then ready for use in flow cytometry or immunofluorescent staining. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact This protocol enables standardisation of parasite sample preparation for downstream assays. Bulk production of material from fewer batches of host iRBC used to grow the parasites reduces sample variability, a limitation of this type of assay. It enables standardised bulk generation of parasite material that can be used across several sites within the grant and avoid site to site variation. Additionally the streamlined process increases overall efficiency. 
 
Title S2 cells expressing different early gametocyte proteins 
Description S2 (Drosophila melanogaster) cells were stably transfected with plasmids for expression of early gametocyte proteins. 
Type Of Material Cell line 
Year Produced 2021 
Provided To Others? No  
Impact Expression of these proteins will be used for immunizations to validate surface expression and immunological potential. 
 
Title Screen for antibody binding to Plasmodium falciparum gametocytes and trophozoites 
Description Pf2004 trophozoites were stained with 1:200 anti-ms glycophorin A and 10 ug/mL DAPI for 30 minutes at 4C. Following the stain, trophozoites were washed in 0.5% BSA in PBS and stained with 1:200 anti-mouse IgG Alexa-Fluor 488 for 30 minutes at 4C. DAPI-stained trophozoites were mixed at a 1:2 ratio with TdTomato-gametocytes and diluted 1:200. 15 uL of parasites were added to each well and mixed with 15 uL plasma diluted 1:10. The samples were incubated for 30 minutes at 4C, washed twice, and acquired on the iQue HT flow cytometer. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact This assay assesses the binding of many samples from malaria-exposed or vaccinated individuals for antibody binding to different parasite stages, allowing us to choose candidates for monoclonal antibody isolation. 
 
Title Bayesian model for parasite kinetics and gametocyte commitment 
Description A bayesian modeling framework was developed based on molecular parasite density data from malaria-naive volunteers who were exposed to P. falciparum by mosquito bite or blood stage parasite inocula. Gametocyte sequestration in the bone marrow and release were modeled based on individual data on ring-stage parasites and gametocytes. 
Type Of Material Computer model/algorithm 
Year Produced 2022 
Provided To Others? No  
Impact The model, that will be submitted for publication later in 2022, provides a critical framework to test the impact of early gametocyte immunity on gametocyte maturation and release. It will thus be of great value in quantifying the epidemiological impact of early gametocyte immunity in naturally exposed populations. 
 
Title Flow cytometry data analysis 
Description Developing a flow cytometry data analysis protocol 
Type Of Material Data analysis technique 
Year Produced 2021 
Provided To Others? No  
Impact Standard method for analysis to enable comparisons across sites and cohorts 
 
Title Set of human samples characterized for late gametocyte immune responses 
Description 2500 samples from a Ugandan cohort were characterised for anti-gametocyte and anti-blood stage immunity. This work so far focused on late-stage gametocyte responses and a panel of known markers of parsite exposure/asexual targets. These analyses were performed by ELISA, surface immunofluorescence assays (SIFA) and Luminex and will be complemented with immune assays for early gametocyte proteins once these proteins become available. 
Type Of Material Data handling & control 
Year Produced 2021 
Provided To Others? No  
Impact This dataset described sample characteristics to allow selection of donors with an interesting phenotype (i.e. high or low gametocyte responses with known (high) malaria exposure). This efforts thus provides an important starting point to select donors for B-cell work and for targeted immune phenotyping. 
 
Description Presentation on early and late stage gametocyte immunity at American Society of Tropical Medicine & Hygiene 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact During a selected oral presentation, I presented a Ugandan cohort that we are using to examine early gametocyte immunity in relation to exposure and subsequent gametocyte production. The talk, followed by a Q&A session, was well appreciated and attended by parasitologists, immunologists and medical specialists interest in malaria and malaria immunity.
Year(s) Of Engagement Activity 2021