This year, the BioMalPar conference took place for the 17th time, but the second time virtually. Three poster presenters stunned their peers with their visually attractive digital posters, presentations and research insights. Out of 90 posters, they received the best poster award by popular vote. Meet the winners!
Sex-specific genetic screens identify hundreds of Plasmodium fertility genes essential for the transmission of malaria parasites
Sexual reproduction of malaria parasites is essential for their transmission by mosquitoes. Biological processes required for Plasmodium fertility include the formation of gametocytes, their transformation into gametes in response to signals from the mosquito, fertilisation in the bloodmeal, meiosis, and the formation of an invasive ookinete. Stage-specific gene expression data suggest that hundreds of parasite genes are uniquely required for sexual reproduction, but previous gene knockout studies have merely scratched the surface of this important aspect of parasite biology. We have mutagenised P. berghei lines that make only fertile male or only fertile female gametocytes, with barcoded PlasmoGEM vectors to screen >1200 targetable genes for sex-specific phenotypes. Our screens identify hundreds of genes with sex-specific roles. The data recapitulate existing knowledge of Plasmodium fertility and assign functions to previously unannotated genes. For the first time, we are gaining an unbiased picture of the molecular mechanisms of Plasmodium fertility at genome-scale, which will lead to a deeper understanding of this novel biology that could serve as targets for transmission blocking drugs or vaccines.
ABCI3 confers pleiotropic drug resistance to antimalarial compounds
Understanding the mechanisms available to the malaria parasite for acquiring multidrug resistance will be important for predicting which genes may become important for clinical resistance in the future. ABC transporters are an important protein family with roles in drug resistance across a variety of organisms, and mutations in PfMDR1 modulate sensitivity to multiple antimalarials. Several other ABC transporters are encoded in the Plasmodium genome, and we have identified mutations in ABCI3 that confer resistance to several experimental antimalarial compounds.
Using in vitro drug selection regimes with a set of four chemically related compounds (SY4, 10, 11, 13), we isolated 12 drug resistant lines that were subjected to whole genome sequencing. All contained either single nucleotide variants (SNVs) or copy number amplifications of abci3. The point mutations were located in or near predicted transmembrane domains, consistent with a role in modifying the substrate specificity of the transporter, and testing of these lines against other compounds chemically-unrelated to the SY series identified a subset to which sensitivity is also affected.
In addition, natural variants of ABCI3 are observed at or near to these putative resistance SNVs, and preliminary evidence indicates differing sensitivities to the SY compounds among field isolates and common lab strains that may be driven by variation in ABCI3.
This work suggests abci3 should be among the genes monitored for changes in prevalence in longitudinal sampling of field isolates.
Characterization of a new malaria vaccine candidate against Plasmodium vivax using genetically modified rodent malaria parasites
Malaria, a mosquito-borne disease caused by Plasmodium parasites, is the most prevalent parasitic infection worldwide. Despite considerable efforts, there is still no effective vaccine against human-infective Plasmodium parasites, of which P. falciparum (Pf) and P. vivax (Pv) are the clinically most significant. Whole-sporozoite (Wsp) vaccines, which induce efficient immune responses against the pre-erythrocytic (PE) stages of Plasmodium parasites, are among the most promising immunization strategies so far. Although most malaria vaccine research has focused on Pf infection, Pv continues to be the most widespread of the human-infective Plasmodium species, imposing significant health and economic burdens on affected countries. Importantly, Pv can originate dormant parasitic liver forms – hypnozoites – which may cause malaria relapses long after mosquito transmission. Recently, our lab developed a new Wsp based on the use of transgenic rodent P. berghei (Pb) parasites as a platform to deliver immunogens of human-infective Plasmodium parasites. Since our in silico studies predict that >60% of CD8+ T cell epitopes encoded in both the Pv and Pb proteomes are shared between these two parasites, we generated a new genetically modified Pb expressing the highly immunogenic circumsporozoite (CS) protein from Pv (PvCS), in addition to its endogenous CS, Pb(PvCS@UIS4), to be used as a vaccine candidate against Pv malaria. Our immunofluorescence microscopy studies confirmed that both the endogenous PbCS and the inserted PvCS are expressed during the PE stages of this transgenic parasite, and that its infectivity is similar to that of its wild-type (WT) counterpart. Specifically, the ability of Pb(PvCS@UIS4) to infect Anopheles stephensi mosquitoes, as measured by the number of oocysts or sporozoites formed, as well as its ability to infect and develop normally in mouse hepatocytes and red blood cells showed no significant differences from those observed for WT parasites. Subsequent studies showed that mice immunization with Pb(PvCS@UIS4) elicits the production of anti-PvCS antibodies that efficiently recognize and bind to Pv sporozoites. Considering the lack of efficient strategies to tackle Pv, this study represents a crucial step on the development of a new Wsp vaccine candidate against this parasite.
Despite the coronavirus pandemic, EMBL’s BioMalPar conference could still take place online. Nearly 400 participants from around the world attended the virtual meeting to share the latest research into Plasmodium, the malaria-causing parasite.
From onsite to digital
There were both benefits and drawbacks to hosting the conference online. On the plus side, the conference’s barrier to entry was significantly reduced, allowing for a higher-than-usual number of participants to attend, from all over the world and at different stages of career. There was more interaction between speakers and the audience and those who would not normally feel comfortable asking a question in a conference hall could do so more comfortably online. The conference was slightly shorter than usual – just two half days – so there were fewer talks overall and no possibility for workshops; however ‘special interest’ lectures were held instead. Poster sessions could still take place with posters remaining online after the session for more leisurely viewing. On a more specific note, Dr Julian Rayner, a member of the BioMalPar Emeritus Steering Committee, remarked that he missed the asparagus stalls in the streets of Heidelberg, where the conference normally takes place. Asparagus or not, it’s remarkable that, in the face of real challenge and uncertainty, the EMBL BioMalPar conference could still take place, now sixteen years in the running.
The conference began with opening remarks from co-chairs Alfred Cortes of ISGlobal in Spain, Silvia Portugal of the Medical University Heidelberg in Germany and Sam Wassmer of the London School of Hygiene and Tropical Medicine in the UK. They emphasised the novelty of the online conference and outlined some ground rules.
Emerging Challenges and New Tools
The first session of the day was on ‘emerging challenges and new tools’ and was chaired by Andy Waters.
Laurent Dembele of the University of Bamako, Ghana, spoke on the subject of artemisinin resistance. Mutations to a protein called Kelch13 prevent the antimalarial from working, allowing the parasite to become dormant in the blood and to ‘reactivate’ when the drug is no longer in the system. Dembele evaluated two antimalarial drugs (KDU691 and GNF179) in vitro which target P14K, a protein that mediates the aforementioned dormancy. He concluded that KDU691 can selectively kill the dormant Plasmodium rings after treatment with artemisinin, but that GNF179 can kill both dormant and non-dormant rings and was active at multiple stages of the malaria parasite life cycle. A combination of artemisinin and GNF179 could mitigate the risk of artemisinin resistance, it was suggested. This has direct applications to SE Asia, where artemisinin resistance is particularly prevalent.
Simone Reber of the Humboldt University of Berlin spoke about her research into Plasmodium tubulin, a potential target for drug development. Tubulin is a ubiquitous protein that controls cell proliferation and so has direct applications to the treatment of malaria, a disease that depends upon cell replication. Simone characterised tubulin in the malaria parasite and considered drugs that could inhibit it and compared Plasmodium tubulin to a similar shaped human tubulin. A concentration of 2.5 micromoles of inhibitor could kill the Plasmodium tubulin but not the human tubulin, for which 20 micromoles were needed. Tubulin is a promising target for malaria treatment but research into it is limited by the protein’s small size. For 150 micrograms of tubulin – in itself, a rather small sample – 5 litres of blood is needed.
Regina Rabinovich of ISGlobal gave her special interest lecture on COVID-19 and its impact on ‘research and beyond’. She took a step back from the specific biology of malaria to consider global public health in general; ‘from the mitotic spindle to global malaria strategy’, as she called it. She offered a concise history of malaria eradication and addressed recent developments in strategy: a shift away from blanket solutions to region-specific programmes. She also shared the broad public health lessons that we can learn from COVID-19, with particular emphasis on accelerating R&D efforts and cultivating a sense of urgency. She concluded with remarks on the potential impact of the coronavirus in Africa both socially and epidemiologically, with reference to data modelling from the WHO and Imperial College London.
Alfred Cortes introduced the second session on ‘parasite biology’.
First to speak was ManojDuraisingh of the Harvard TH Chan School of Public Health. He spoke on how the P. falciparum egresses from red blood cells. He detailed that the PfPP1 protein is essential for such egress and that it coordinates multiple signals early in egress. He also outlined the role for Hect E3 ligase in egrees and that PfPP1 regulated cGMP levels through PLT-GCalpha.
MathieuBrochet of the University of Geneva spoke on his work into the Plasmodium protein, PKG. This protein was previously known to control calcium signals called Ca2+ which, in turn, are critical for Plasmodium life cycle stages in the human and mosquito, namely erythrocytic egress and gametocyte development. How PKG influenced these Ca2+ signals was previously unknown, however, so his research sought to identify a partner protein. He identified ICM1 as this partner protein, finding that when ICM1 was distributed, so was egress, invasion and Ca2+ mobilisation.
FranckDumetz of the University of Cambridge spoke about his investigations into the presence of ‘G4’ proteins in the P. falciparum genome and whether they play a role in post-transcriptional regulation. He first identified 681 ‘rG4s’ in the transcriptome and used both in vitro and in vivo approaches to find that rG4s can have a regressive impact on translation when they appear on the transcribed strand of a gene. He continued to suggest that there may be novel RNA-binding proteins in Plasmodium that are involved in regulating rG4 motifs.
Venessa Zuzarte-Luis shared her research into why Plasmodium replication in the liver of mammals is particularly high, producing tens of thousands of parasites, compared to replication in reptile and avian species, which only produce dozens of parasites. Vanessa states that this disparity is caused by the parasite’s utilisation of liver-specific methionine adenosyltransferase enzyme (MAT1), and the capacity of that enzyme to generate unlimited amounts of S-adenosylmethionine (SAM). SAM is capable of reverting the impairment of infection, suggesting that the parasites use this abundant resource.
“Omics” Approaches and Evolution
After a poster session on Slack, Julian Rayner began the third session on “omics” approaches and evolution.
Franziska Hentzschel of the University of Glasgow presented her research into the parasite-host interplay in vivo at the single-cell level. She used flow cytometry and single-cell RNAseq to characterise parasite and host cells in circulating blood, bone marrow and the spleen of P. berghei-infected mice. She found that the spleen was preferred as the extravascular site for Plasmodium reservoirs, with the earliest ring stages enriched in putative reticulocytes found there.
LianneLansink of the Queensland University of Technology and the University of Melbourne detailed her research into how the malaria parasite adapts in the human host to changes in the environment. She created an environment of lipopolysaccharide, a condition which causes low blood pressure. She reported that this impaired the maturation of the parasites in vivo. The condition also affected parasitic gene expression; ribosome genes were down-regulated and other genes were perturbed in single parasites in a life-stage dependent manner.
Epidemiology and Surveillance
The second day of the BioMalPar conference began with a virtual pub quiz. Sam Wassmer then introduced the fourth virtual session of the event: ‘epidemiology and surveillance’.
ChrisDrakeley of the London School of Hygiene and Tropical Medicine gave a talk on the use of serology in understanding malaria transmission dynamics. He outlined several use cases of serology in malaria-endemic regions and emphasised the advancements in serological technologies; multiplex bead assays and protein/peptide microarrays allow for the assessment of proteome-wide immune responses which allows for greater granularity of analysis, looking at specific antigens or antigen combinations that cause specific immune responses.
MarioRecker of the University of Exeter spoke about understanding individual-level variations in malaria susceptibility and acquisition of clinical protection. His study sought to identify robust inferences of individual-level protection or susceptibility to malaria by analysing a longitudinal systems-immunological cohort study of Junju, a region of Kenya. He found that one of the most important risk factors to malaria is the number of previously experienced episodes which correlates positively with probability and number of clinical episodes during the upcoming transmission season. He also found that birth year can also have a significant and lasting impact on a child’s susceptibility to clinical malaria, due to the child’s exposure to Plasmodium early on in life.
ManuelaCarrasquilla spoke about her research into genome sequencing in Colombia to determine the history of P. falciparum. She analysed 151 clinical samples of the parasite collected in a regional hotspot during a 2016 outbreak linked to mining activities. She determined that the Colombian P. falciparum populations show high levels of genetic structuring and confirmed the presence of five major parasite lineages overlapping in time and space which have been shown to have important functional variation, including drug-resistance genes. Then, combining epidemiological data over a two-year period, she found that almost a third of infections show evidence of shared recent common ancestry, suggesting that increasing mining activities created an opportunity for polyclonal infections and outcrossing.
SimonKigozi of the London School of Hygiene and Tropical Medicines presented the results of his analysis of outpatient surveillance data from four health facilities in Uganda. The malaria burden had shifted from younger to older individuals as a result of malaria control methods including long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS).
Pathogenesis and Immunology
SamWassmer began the fifth virtual session of the conference on ‘pathogenesis & immunology’.
BrittanyRiggle of the NIH was first to speak on the subject of pathogenesis and her work into CD8+ T cells as a potential avenue for treating cerebral malaria. Using multiplex immunohistochemistry in post-mortem brain samples from children with or without cerebral malaria and with HIV. Diagnosis of cerebral malaria increased the number of CD3+CD8+ T cells engaging the cerebrovasculature. HIV co-infection further increased this engagement. Riggle suggests that CD3+CD8+ T cells could be a promising target for adjunctive therapy for cerebral malaria.
HannahFleckenstein of Heidelberg University Hospital spoke about her research into how P. falciparum malaria parasites persist at low parasitemia for months in the human host in the absence of the Anopheles mosquito vector, a poorly understood phenomenon. In the dry season in Mali, the parasites spent longer in circulation before adhering to the endothelium. This adhesion is mediated by knob-like structures on the surface of infected red blood cells which allows for the avoidance of splenic clearance. She found that, in dry season parasites, decreased adhesion resulted in higher levels of splenic clearance which was sufficient to maintain low parasitemia.
Speaking on the subject of monoclonal antibodies, LawrenceWang of the NIH presented his findings into a sporozoite-neutralising monoclonal antibody called L9. The mAb could immobilise sporozoites in the skin and the liver and cause a phenomenon called ‘dotty death’. When asked why monoclonal antibodies would be preferred over mass-market drugs, which are cheaper and less laborious to produce, Lawrence responded by saying that some mAbs can offer potentially life-long protection and are a halfway house between a drug and a vaccine.
EmiliePollenus of KU Leuven spoke about the role of CCR2 in the resolution of MA-ARDS (malaria-associated acute respiratory distress syndrome). This condition is one of the health complications that malaria can cause, contributing to its 400,000 annual death toll. Those with the condition who do not have access to mechanical ventilation have an 80% risk of death, while those with it have a 10-40% risk. Pollenus found that the number of CCR2 monocytes increased during resolution after treatment with antimalarial medicine. In mice where the CCR2 protein was silenced – known as CCR2 KO (knock out) mice – there was a reduction in the number of inflammatory monocytes in the lungs and the spleen, however, this did not affect the resolution of the alveolar edema. Eosinophils – a type of white blood cell – completely disappeared from the lungs upon infection and reappeared with resolution but not in CCR2 KO mice.
CarolaSchaefer of the Seattle Children’s Research Institute spoke about creating a laboratory model for P. vivax infection to test interventions against the disease. Using the P.vivax liver humanised mouse model, with the addition of human reticulocytes, parasites were able to invade and replicate Schaefer could also observe gametocytes that were transmitted to mosquitoes in preliminary experiments.. She then tested the in vivo model with P. vivax Duffy Binding Protein (PvDBP) antibodies, an antibody that could potentially inhibit parasite invasion of red blood cells. 95% protection was observed, providing evidence that PvDBP is a promising target for future therapies.
SilviaPortugal opened the final virtual session of the conference on transmission biology.
DennisKlug of Inserm in France presented his research into the function of the protein TEP12 in antiparasitic immunity in Anopheles mosquitoes. When the protein was silenced, there was an increased parasitic load in P. berghei infections in An. gambiae and An. stephensi mosquitoes. However, there was a lower parasitic load with P. falciparum infection, suggesting an unknown adaptation of this parasite species.
AureliaBalestra of the University of Geneva discussed her identification of CDK-related kinase 5 (CRK5) as a critical regulator of atypical mitosis in the gametogony[KC1] which is required for mosquito transmission. This kinase interacts with a single Plasmodium-specific cyclin (SOC2), suggesting this cyclin/CDK pair controls DNA replication and M-phase progression.
InesBento of the University of Texas Southwestern Medical Centre presented her findings into mosquito-human sporozoite transmission. She found that sporozoites in the salivary glands of the mosquito are in synchrony with the vector’s behaviour, leading to lower parasitic load in the day compared to the night, when the mosquito is more prone to bite.
Bento’s talk concluded the session and was the last talk of the conference. AndyWaters then gave a memorial presentation on Shahid Khan, a malariologist who passed away in late 2019. He began his career with a degree in Parasitology from the University of Glasgow and then worked as a technician at the University of Oxford. He completed a PhD at Cambridge and a Postdoc at NIMR in London. Tributes were made to Shahid by his colleagues; he was described as the ‘sunshine’ of the laboratory and recently wrote a children’s bedtime story entitled ‘There’s a mosquito in my room’, which is available to download here.
The conference was formally closed with remarks from AndyWaters and JulianRayner. They called the virtual meeting a successful and ‘wonderful experiment’.
About the author
I’m Thomas Locke, the Editor of Fight Malaria, a website that aims to distil the science of malaria and make it more accessible to the public. Every week, we summarise some of the malaria research highlights in a sixty-second podcast called ‘Malaria Minute’ and also interview various stakeholders in the fight against malaria. You can learn more about the initiative here. I was fortunate to work as an ‘Event Reporter’ for this year’s BioMalPar and offer a summary below. I’d like to offer thanks to EMBL for covering the cost of my ticket.