Structural basis of malaria transmission blockade by a monoclonal antibody to gamete fusogen HAP2.

HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.

Dissection-independent production of Plasmodium sporozoites from whole mosquitoes.

Progress towards a protective vaccine against malaria remains slow. To date, only limited protection has been routinely achieved following immunisation with either whole-parasite (sporozoite) or subunit-based vaccines. One major roadblock to vaccine progress, and to pre-erythrocytic parasite biology in general, is the continued reliance on manual salivary gland dissection for sporozoite isolation from infected mosquitoes. Here, we report development of a multi-step method, based on batch processing of homogenised whole mosquitoes, slurry, and density-gradient filtration, which combined with free-flow electrophoresis rapidly produces a pure, infective sporozoite inoculum. Human-infective Plasmodium falciparum and rodent-infective Plasmodium berghei sporozoites produced in this way are two- to threefold more infective than salivary gland dissection sporozoites in in vitro hepatocyte infection assays. In an in vivo rodent malaria model, the same P. berghei sporozoites confer sterile protection from mosquito-bite challenge when immunisation is delivered intravenously or 60-70% protection when delivered intramuscularly. By improving purity, infectivity, and immunogenicity, this method represents a key advancement in capacity to produce research-grade sporozoites, which should impact delivery of a whole-parasite based malaria vaccine at scale in the future.

Detection of Plasmodium falciparum in laboratory-reared and naturally infected wild mosquitoes using near-infrared spectroscopy.

There is an urgent need for high throughput, affordable methods of detecting pathogens inside insect vectors to facilitate surveillance. Near-infrared spectroscopy (NIRS) has shown promise to detect arbovirus and malaria in the laboratory but has not been evaluated in field conditions. Here we investigate the ability of NIRS to identify Plasmodium falciparum in Anopheles coluzzii mosquitoes. NIRS models trained on laboratory-reared mosquitoes infected with wild malaria parasites can detect the parasite in comparable mosquitoes with moderate accuracy though fails to detect oocysts or sporozoites in naturally infected field caught mosquitoes. Models trained on field mosquitoes were unable to predict the infection status of other field mosquitoes. Restricting analyses to mosquitoes of uninfectious and highly-infectious status did improve predictions suggesting sensitivity and specificity may be better in mosquitoes with higher numbers of parasites. Detection of infection appears restricted to homogenous groups of mosquitoes diminishing NIRS utility for detecting malaria within mosquitoes.

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.

Male-Specific Protein Disulphide Isomerase Function is Essential for Plasmodium Transmission and a Vulnerable Target for Intervention.

Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. Lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface-expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first evidence that PDI function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking interventions.

Synergy in anti-malarial pre-erythrocytic and transmission-blocking antibodies is achieved by reducing parasite density.

Anti-malarial pre-erythrocytic vaccines (PEV) target transmission by inhibiting human infection but are currently partially protective. It has been posited, but never demonstrated, that co-administering transmission-blocking vaccines (TBV) would enhance malaria control. We hypothesized a mechanism that TBV could reduce parasite density in the mosquito salivary glands, thereby enhancing PEV efficacy. This was tested using a multigenerational population assay, passaging Plasmodium berghei to Anopheles stephensi mosquitoes. A combined efficacy of 90.8% (86.7-94.2%) was observed in the PEV +TBV antibody group, higher than the estimated efficacy of 83.3% (95% CrI 79.1-87.0%) if the two antibodies acted independently. Higher PEV efficacy at lower mosquito parasite loads was observed, comprising the first direct evidence that co-administering anti-sporozoite and anti-transmission interventions act synergistically, enhancing PEV efficacy across a range of TBV doses and transmission intensities. Combining partially effective vaccines of differing anti-parasitic classes is a pragmatic, powerful way to accelerate malaria elimination efforts.

Adenovirus-prime and baculovirus-boost heterologous immunization achieves sterile protection against malaria sporozoite challenge in a murine model.

With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.

Targeting the Conserved Fusion Loop of HAP2 Inhibits the Transmission of Plasmodium berghei and falciparum.

Inhibiting transmission of Plasmodium is a central strategy in malarial eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. The lack of a structure or known molecular function of current anti-malarial vaccine targets has previously been a hindrance in the development of transmission-blocking vaccines. Structure/function studies have indicated that the conserved gamete membrane fusion protein HAP2 is a class II viral fusion protein. Here, we demonstrate that targeting a function-critical site of the fusion/cd loop with species-specific antibodies reduces Plasmodium berghei transmission in vivo by 58.9% and in vitro fertilization by up to 89.9%. A corresponding reduction in P. falciparum transmission (75.5%/36.4% reductions in intensity/prevalence) is observed in complimentary field studies. These results emphasize conserved mechanisms of fusion in Apicomplexa, while highlighting an approach to design future anti-malarial transmission-blocking vaccines.

A novel model fitted to multiple life stages of malaria for assessing efficacy of transmission-blocking interventions.

BACKGROUND: Transmission-blocking interventions (TBIs) aim to eliminate malaria by reducing transmission of the parasite between the host and the invertebrate vector. TBIs include transmission-blocking drugs and vaccines that, when given to humans, are taken up by mosquitoes and inhibit parasitic development within the vector. Accurate methodologies are key to assess TBI efficacy to ensure that only the most potent candidates progress to expensive and time-consuming clinical trials. Measuring intervention efficacy can be problematic because there is substantial variation in the number of parasites in both the host and vector populations, which can impact transmission even in laboratory settings. METHODS: A statistically robust empirical method is introduced for estimating intervention efficacy from standardised population assay experiments. This method will be more reliable than simple summary statistics as it captures changes in parasite density in different life-stages. It also allows efficacy estimates at a finer resolution than previous methods enabling the impact of the intervention over successive generations to be tracked. A major advantage of the new methodology is that it makes no assumptions on the population dynamics of infection. This enables both host-to-vector and vector-to-host transmission to be density-dependent (or other) processes and generates easy-to-understand estimates of intervention efficacy. RESULTS: This method increases the precision of intervention efficacy estimates and demonstrates that relying on changes in infection prevalence (the proportion of infected hosts) alone may be insufficient to capture the impact of TBIs, which also suppress parasite density in secondarily infected hosts. CONCLUSIONS: The method indicates that potentially useful, partially effective TBIs may require multiple infection cycles before substantial reductions in prevalence are observed, despite more rapidly suppressing parasite density. Accurate models to quantify efficacy will have important implications for understanding how TBI candidates might perform in field situations and how they should be evaluated in clinical trials.

Probability of Transmission of Malaria from Mosquito to Human Is Regulated by Mosquito Parasite Density in Naïve and Vaccinated Hosts.

Over a century since Ronald Ross discovered that malaria is caused by the bite of an infectious mosquito it is still unclear how the number of parasites injected influences disease transmission. Currently it is assumed that all mosquitoes with salivary gland sporozoites are equally infectious irrespective of the number of parasites they harbour, though this has never been rigorously tested. Here we analyse >1000 experimental infections of humans and mice and demonstrate a dose-dependency for probability of infection and the length of the host pre-patent period. Mosquitoes with a higher numbers of sporozoites in their salivary glands following blood-feeding are more likely to have caused infection (and have done so quicker) than mosquitoes with fewer parasites. A similar dose response for the probability of infection was seen for humans given a pre-erythrocytic vaccine candidate targeting circumsporozoite protein (CSP), and in mice with and without transfusion of anti-CSP antibodies. These interventions prevented infection more efficiently from bites made by mosquitoes with fewer parasites. The importance of parasite number has widespread implications across malariology, ranging from our basic understanding of the parasite, how vaccines are evaluated and the way in which transmission should be measured in the field. It also provides direct evidence for why the only registered malaria vaccine RTS,S was partially effective in recent clinical trials.

A novel multiple-stage antimalarial agent that inhibits protein synthesis.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.