Detailing organelle division and segregation in Plasmodium falciparum.

The malaria causing parasite, P. falciparum, replicates through a tightly orchestrated process termed schizogony, where approximately 32 daughter parasites are formed in a single infected red blood cell and thousands of daughter cells in mosquito- or liver-stages. One-per-cell organelles, such as the mitochondrion and apicoplast, need to be properly divided and segregated to ensure a complete set of organelles per daughter parasites. Although this is highly essential, details about the processes and mechanisms involved remain unknown. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood- and mosquito stages. Using high-resolution 3D-imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during the last stage of schizogony. Analysis of focused ion beam scanning electron microscopy (FIB-SEM) data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques (CPs) in apicoplast segregation. These observations form the foundation for a new detailed model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the mechanisms of organelle division and segregation.

Pfs230 Domain 7 is targeted by a potent malaria transmission-blocking monoclonal antibody.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, mAb 18F25.2a, using a CRISPR/Cas9-based hybridoma engineering method. This subclass-switched mAb 18F25.2a induced lysis of female gametes in vitro. Importantly, mAb 18F25.2a potently reduced P. falciparum infection of Anopheles stephensi mosquitoes in a complement-dependent manner, as assessed by standard membrane feeding assays. Together, our data identify Pfs230 Domain 7 as target for transmission-blocking antibodies and provide a strong incentive to study domains outside Pfs230Pro-D1 as TBV candidates.

Rapid profiling of Plasmodium parasites from genome sequences to assist malaria control.

BACKGROUND: Malaria continues to be a major threat to global public health. Whole genome sequencing (WGS) of the underlying Plasmodium parasites has provided insights into the genomic epidemiology of malaria. Genome sequencing is rapidly gaining traction as a diagnostic and surveillance tool for clinical settings, where the profiling of co-infections, identification of imported malaria parasites, and detection of drug resistance are crucial for infection control and disease elimination. To support this informatically, we have developed the Malaria-Profiler tool, which rapidly (within minutes) predicts Plasmodium species, geographical source, and resistance to antimalarial drugs directly from WGS data. RESULTS: The online and command line versions of Malaria-Profiler detect ~ 250 markers from genome sequences covering Plasmodium speciation, likely geographical source, and resistance to chloroquine, sulfadoxine-pyrimethamine (SP), and other anti-malarial drugs for P. falciparum, but also providing mutations for orthologous resistance genes in other species. The predictive performance of the mutation library was assessed using 9321 clinical isolates with WGS and geographical data, with most being single-species infections (P. falciparum 7152/7462, P. vivax 1502/1661, P. knowlesi 143/151, P. malariae 18/18, P. ovale ssp. 5/5), but co-infections were identified (456/9321; 4.8%). The accuracy of the predicted geographical profiles was high to both continental (96.1%) and regional levels (94.6%). For P. falciparum, markers were identified for resistance to chloroquine (49.2%; regional range: 24.5% to 100%), sulfadoxine (83.3%; 35.4- 90.5%), pyrimethamine (85.4%; 80.0-100%) and combined SP (77.4%). Markers associated with the partial resistance of artemisinin were found in WGS from isolates sourced from Southeast Asia (30.6%). CONCLUSIONS: Malaria-Profiler is a user-friendly tool that can rapidly and accurately predict the geographical regional source and anti-malarial drug resistance profiles across large numbers of samples with WGS data. The software is flexible with modifiable bioinformatic pipelines. For example, it is possible to select the sequencing platform, display specific variants, and customise the format of outputs. With the increasing application of next-generation sequencing platforms on Plasmodium DNA, Malaria-Profiler has the potential to be integrated into point-of-care and surveillance settings, thereby assisting malaria control. Malaria-Profiler is available online (bioinformatics.lshtm.ac.uk/malaria-profiler) and as standalone software ( https://github.com/jodyphelan/malaria-profiler ).

Whole sporozoite immunization with Plasmodium falciparum strain NF135 in a randomized trial.

BACKGROUND: Whole sporozoite immunization under chemoprophylaxis (CPS regime) induces long-lasting sterile homologous protection in the controlled human malaria infection model using Plasmodium falciparum strain NF54. The relative proficiency of liver-stage parasite development may be an important factor determining immunization efficacy. Previous studies show that Plasmodium falciparum strain NF135 produces relatively high numbers of large liver-stage schizonts in vitro. Here, we evaluate this strain for use in CPS immunization regimes. METHODS: In a partially randomized, open-label study conducted at the Radboudumc, Nijmegen, the Netherlands, healthy, malaria-naïve adults were immunized by three rounds of fifteen or five NF135-infected mosquito bites under mefloquine prophylaxis (cohort A) or fifteen NF135-infected mosquito bites and presumptive treatment with artemether/lumefantrine (cohort B). Cohort A participants were exposed to a homologous challenge 19 weeks after immunization. The primary objective of the study was to evaluate the safety and tolerability of CPS immunizations with NF135. RESULTS: Relatively high liver-to-blood inocula were observed during immunization with NF135 in both cohorts. Eighteen of 30 (60%) high-dose participants and 3/10 (30%) low-dose participants experienced grade 3 adverse events 7 to 21 days following their first immunization. All cohort A participants and two participants in cohort B developed breakthrough blood-stage malaria infections during immunizations requiring rescue treatment. The resulting compromised immunizations induced modest sterile protection against homologous challenge in cohort A (5/17; 29%). CONCLUSIONS: These CPS regimes using NF135 were relatively poorly tolerated and frequently required rescue treatment, thereby compromising immunization efficiency and protective efficacy. Consequently, the full potential of NF135 sporozoites for induction of immune protection remains inconclusive. Nonetheless, the high liver-stage burden achieved by this strain highlights it as an interesting potential candidate for novel whole sporozoite immunization approaches. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov under identifier NCT03813108.

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 µg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230.

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses.

Malaria transmission-blocking vaccines (TBVs) aim to elicit human antibodies that inhibit sporogonic development of Plasmodium falciparum in mosquitoes, thereby preventing onward transmission. Pfs48/45 is a leading clinical TBV candidate antigen and is recognized by the most potent transmission-blocking monoclonal antibody (mAb) yet described; still, clinical development of Pfs48/45 antigens has been hindered, largely by its poor biochemical characteristics. Here, we used structure-based computational approaches to design Pfs48/45 antigens stabilized in the conformation recognized by the most potently inhibitory mAb, achieving >25°C higher thermostability compared with the wild-type protein. Antibodies elicited in mice immunized with these engineered antigens displayed on liposome-based or protein nanoparticle-based vaccine platforms exhibited 1-2 orders of magnitude superior transmission-reducing activity, compared with immunogens bearing the wild-type antigen, driven by improved antibody quality. Our data provide the founding principles for using molecular stabilization solely from antibody structure-function information to drive improved immune responses against a parasitic vaccine target.

Safety, tolerability, and Plasmodium falciparum transmission-reducing activity of monoclonal antibody TB31F: a single-centre, open-label, first-in-human, dose-escalation, phase 1 trial in healthy malaria-naive adults.

BACKGROUND: Malaria elimination requires interruption of the highly efficient transmission of Plasmodium parasites by mosquitoes. TB31F is a humanised monoclonal antibody that binds the gamete surface protein Pfs48/45 and inhibits fertilisation, thereby preventing further parasite development in the mosquito midgut and onward transmission. We aimed to evaluate the safety and efficacy of TB31F in malaria-naive participants. METHODS: In this open-label, first-in-human, dose-escalation, phase 1 clinical trial, healthy, malaria-naive, adult participants were administered a single intravenous dose of 0·1, 1, 3, or 10 mg/kg TB31F or a subcutaneous dose of 100 mg TB31F, and monitored until day 84 after administration at a single centre in the Netherlands. The primary outcome was the frequency and magnitude of adverse events. Additionally, TB31F serum concentrations were measured by ELISA. Transmission-reducing activity (TRA) of participant sera was assessed by standard membrane feeding assays with Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. The trial is registered with Clinicaltrials.gov, NCT04238689. FINDINGS: Between Feb 17 and Dec 10, 2020, 25 participants were enrolled and sequentially assigned to each dose (n=5 per group). No serious or severe adverse events occurred. In total, 33 grade 1 and six grade 2 related adverse events occurred in 20 (80%) of 25 participants across all groups. Serum of all participants administered 1 mg/kg, 3 mg/kg, or 10 mg/kg TB31F intravenously had more than 80% TRA for 28 days or more, 56 days or more, and 84 days or more, respectively. The TB31F serum concentration reaching 80% TRA was 2·1 µg/mL (95% CI 1·9-2·3). Extrapolating the duration of TRA from antibody kinetics suggests more than 80% TRA is maintained for 160 days (95% CI 136-193) following a single intravenous 10 mg/kg dose. INTERPRETATION: TB31F is a well tolerated and highly potent monoclonal antibody capable of completely blocking transmission of P falciparum parasites from humans to mosquitoes. In areas of seasonal transmission, a single dose might cover an entire malaria season. FUNDING: PATH's Malaria Vaccine Initiative.

Heterologous Expression and Evaluation of Novel Plasmodium falciparum Transmission Blocking Vaccine Candidates.

Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodium falciparum development in the mosquito midgut and thereby prevent onward malaria transmission. A limited number of TBV candidates have been identified and only three (Pfs25, Pfs230 and Pfs48/45) have entered clinical testing. While one of these candidates may emerge as a highly potent TBV candidate, it is premature to determine if they will generate sufficiently potent and sustained responses. It is therefore important to explore novel candidate antigens. We recently analyzed sera from naturally exposed individuals and found that the presence and/or intensity of antibodies against 12 novel putative surface expressed gametocyte antigens was associated with transmission reducing activity. In this study, protein fragments of these novel TBV candidates were designed and heterologously expressed in Drosophila melanogaster S2 cells and Lactococcus lactis. Eleven protein fragments, covering seven TBV candidates, were successfully produced. All tested antigens were recognized by antibodies from individuals living in malaria-endemic areas, indicating that native epitopes are present. All antigens induced antigen-specific antibody responses in mice. Two antigens induced antibodies that recognized a native protein in gametocyte extract, and antibodies elicited by four antigens recognized whole gametocytes. In particular, we found that antigen Pf3D7_0305300, a putative transporter, is abundantly expressed on the surface of gametocytes. However, none of the seven novel TBV candidates expressed here induced an antibody response that reduced parasite development in the mosquito midgut as assessed in the standard membrane feeding assay. Altogether, the antigen fragments used in this study did not prove to be promising transmission blocking vaccine constructs, but led to the identification of two gametocyte surface proteins that may provide new leads for studying gametocyte biology.

The effect of anticoagulants in blood collection tubes on Plasmodium falciparum transmission in direct membrane feeding assays.

BACKGROUND: Direct membrane feeding assays assess the transmission potential of malaria-infected individuals using whole blood collected in anticoagulant vacutainers. METHODS: The potential inhibitory effect of four commonly used anticoagulants on gametocyte infectivity to mosquitoes was assessed in standard membrane feeding assays with cultured Plasmodium falciparum. RESULTS: Infection burden in mosquitoes was significantly reduced when blood was collected in sodium citrate and EDTA. Transmission was highest when blood was collected in lithium heparin and sodium heparin, although a concentration-dependent inhibition of mosquito infection was also observed. CONCLUSIONS: Although anticoagulants can reduce transmission efficiency, lithium heparin and sodium heparin are the best anticoagulants for evaluating malaria transmission.

Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine.

The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230-Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM, elicited functional antibodies that reduced transmission to mosquitoes and sporozoite invasion of human hepatocytes. Altogether, our data support manufacture and clinical evaluation of ProC6C as a multistage malaria-vaccine candidate.

A portfolio of geographically distinct laboratory-adapted Plasmodium falciparum clones with consistent infection rates in Anopheles mosquitoes.

BACKGROUND: The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations. METHODS: Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. RESULTS: Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). CONCLUSIONS: These new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

CRISPR/Cas9-engineered inducible gametocyte producer lines as a valuable tool for Plasmodium falciparum malaria transmission research.

The malaria parasite Plasmodium falciparum replicates inside erythrocytes in the blood of infected humans. During each replication cycle, a small proportion of parasites commits to sexual development and differentiates into gametocytes, which are essential for parasite transmission via the mosquito vector. Detailed molecular investigation of gametocyte biology and transmission has been hampered by difficulties in generating large numbers of these highly specialised cells. Here, we engineer P. falciparum NF54 inducible gametocyte producer (iGP) lines for the routine mass production of synchronous gametocytes via conditional overexpression of the sexual commitment factor GDV1. NF54/iGP lines consistently achieve sexual commitment rates of 75% and produce viable gametocytes that are transmissible by mosquitoes. We also demonstrate that further genetic engineering of NF54/iGP parasites is a valuable tool for the targeted exploration of gametocyte biology. In summary, we believe the iGP approach developed here will greatly expedite basic and applied malaria transmission stage research.

Monoclonal antibodies block transmission of genetically diverse Plasmodium falciparum strains to mosquitoes.

Malaria parasite transmission to mosquitoes relies on the uptake of sexual stage parasites during a blood meal and subsequent formation of oocysts on the mosquito midgut wall. Transmission-blocking vaccines (TBVs) and monoclonal antibodies (mAbs) target sexual stage antigens to interrupt human-to-mosquito transmission and may form important tools for malaria elimination. Although most epitopes of these antigens are considered highly conserved, little is known about the impact of natural genetic diversity on the functional activity of transmission-blocking antibodies. Here we measured the efficacy of three mAbs against leading TBV candidates (Pfs48/45, Pfs25 and Pfs230) in transmission assays with parasites from naturally infected donors compared to their efficacy against the strain they were raised against (NF54). Transmission-reducing activity (TRA) was measured as reduction in mean oocyst intensity. mAb 45.1 (α-Pfs48/45) and mAb 4B7 (α-Pfs25) reduced transmission of field parasites from almost all donors with IC80 values similar to NF54. Sequencing of oocysts that survived high mAb concentrations did not suggest enrichment of escape genotypes. mAb 2A2 (α-Pfs230) only reduced transmission of parasites from a minority of the donors, suggesting that it targets a non-conserved epitope. Using six laboratory-adapted strains, we revealed that mutations in one Pfs230 domain correlate with mAb gamete surface binding and functional TRA. Our findings demonstrate that, despite the conserved nature of sexual stage antigens, minor sequence variation can significantly impact the efficacy of transmission-blocking mAbs. Since mAb 45.1 shows high potency against genetically diverse strains, our findings support its further clinical development and may inform Pfs48/45 vaccine design.

Maintaining Plasmodium falciparum gametocyte infectivity during blood collection and transport for mosquito feeding assays in the field.

BACKGROUND: Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. METHODS: The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. RESULTS: Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. CONCLUSIONS: This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings.

Zonal human hepatocytes are differentially permissive to Plasmodium falciparum malaria parasites.

Plasmodium falciparum (Pf) is a major cause of human malaria and is transmitted by infected Anopheles mosquitoes. The initial asymptomatic infection is characterized by parasite invasion of hepatocytes, followed by massive replication generating schizonts with blood-infective merozoites. Hepatocytes can be categorized by their zonal location and metabolic functions within a liver lobule. To understand specific host conditions that affect infectivity, we studied Pf parasite liver stage development in relation to the metabolic heterogeneity of fresh human hepatocytes. We found selective preference of different Pf strains for a minority of hepatocytes, which are characterized by the particular presence of glutamine synthetase (hGS). Schizont growth is significantly enhanced by hGS uptake early in development, showcasing a novel import system. In conclusion, Pf development is strongly determined by the differential metabolic status in hepatocyte subtypes. These findings underscore the importance of detailed understanding of hepatocyte host-Pf interactions and may delineate novel pathways for intervention strategies.

A Randomized Clinical Trial to Compare Plasmodium falciparum Gametocytemia and Infectivity After Blood-Stage or Mosquito Bite-Induced Controlled Malaria Infection.

BACKGROUND: For malaria elimination efforts, it is important to better understand parasite transmission to mosquitoes and develop models for early-clinical evaluation of transmission-blocking interventions. METHODS: In a randomized open-label trial, 24 participants were infected by bites from Plasmodium falciparum 3D7-infected mosquitoes (mosquito bite [MB]; n = 12) or by induced blood-stage malaria (IBSM) with the same parasite line (n = 12). After subcurative piperaquine treatment, asexual parasite and gametocytes kinetics were assessed, and mosquito feeding experiments were performed. RESULTS: Study procedures were well tolerated. The median peak gametocyte density was 1304/mL (interquartile range, 308-1607/mL) after IBSM, compared with 14/mL (10-64/mL) after MB inoculation (P < .001), despite similar peak asexual parasite densities (P = .48). Peak gametocyte density was correlated with preceding pfap2-g transcripts, indicative of gametocyte commitment (ρ = 0.62; P = .002). Direct feeding assays resulted in mosquito infections from 9 of 12 participants after IBSM versus 0 of 12 after MB inoculation (P < .001). CONCLUSIONS: We observed a striking effect of inoculation method on gametocyte production, suggesting higher gametocyte commitment after IBSM. Our direct comparison of MB and IBSM establishes the controlled human malaria infection transmission model, using intravenous administration of P. falciparum-infected erythrocytes as a model for early-clinical evaluation of interventions that aim to interrupt malaria transmission. CLINICAL TRIAL REGISTRATION: NCT03454048.

A mosquito feeding assay to examine Plasmodium transmission to mosquitoes using small blood volumes in 3D printed nano-feeders.

BACKGROUND: To understand the dynamics of malaria transmission, membrane feeding assays with glass feeders are used to assess the transmission potential of malaria infected individuals to mosquitoes. However, in some circumstances, use of these assays is hindered by both the blood volume requirement and the availability of fragile, specially crafted glass feeders. 3D printed plastic feeders that require very small volumes of blood would thus expand the utility of membrane feeding assays. METHODS: Using two 3D printing production methods, MultiJet (MJ) and Digital Light Processing (DLP), we developed a plastic version of the most commonly used standard glass feeder (the mini-feeder) with an improved design, and also a smaller feeder requiring only 60 µl of blood (the nano-feeder). Performance of the 3D printed feeders was compared to standard glass mini-feeders by assessing infectivity of gametocytes to mosquitoes in standard membrane feeding assays with laboratory reared Anopheles stephensi mosquitoes and cultured Plasmodium falciparum gametocytes. In addition, the optimum number of mosquitoes that can feed on the nano-feeder was determined by evaluating fully fed mosquitoes visually and by assessing blood- meal volume with a colorimetric haemoglobin assay. RESULTS: The 3D printing methods allowed quick and inexpensive production of durable feeders. Infectivity of gametocytes to mosquitoes was comparable for MJ and DLP 3D printed feeders and glass feeders, and the performance of the 3D printed feeders was not influenced by repeated washing with bleach. There was no loss in transmission efficiency when the feeder size was reduced from mini-feeder to nano-feeder, and blood-meal volume assessment indicated ~10 An. stephensi mosquitoes can take a full blood-meal (median volume 3.44 µl) on a nano-feeder. CONCLUSIONS: Here we present 3D printed mini- and nano-feeders with comparable performance to the currently used glass mini-feeders. These feeders do not require specialized glass craftsmanship, making them easily accessible. Moreover, the smaller nano-feeders will enable evaluation of smaller blood volumes that can be collected from finger prick, thus expanding the utility of membrane feeding assays and facilitating a more thorough evaluation of the human infectious reservoir for malaria.

A double-blind, placebo-controlled phase 1/2a trial of the genetically attenuated malaria vaccine PfSPZ-GA1.

Immunization with attenuated Plasmodium sporozoites can induce protection against malaria infection, as shown by Plasmodium falciparum (Pf) sporozoites attenuated by radiation in multiple clinical trials. As alternative attenuation strategy with a more homogeneous population of Pf sporozoites (PfSPZ), genetically engineered Plasmodium berghei sporozoites (SPZ) lacking the genes b9 and slarp induced sterile protection against malaria in mice. Consequently, PfSPZ-GA1 Vaccine, a Pf identical double knockout (Pf∆b9∆slarp), was generated as a genetically attenuated malaria parasite vaccine and tested for safety, immunogenicity, and preliminary efficacy in malaria-naïve Dutch volunteers. Dose-escalation immunizations up to 9.0 × 105 PfSPZ of PfSPZ-GA1 Vaccine were well tolerated without breakthrough blood-stage infection. Subsequently, groups of volunteers were immunized three times by direct venous inoculation with cryopreserved PfSPZ-GA1 Vaccine (9.0 × 105 or 4.5 × 105 PfSPZ, N = 13 each), PfSPZ Vaccine (radiation-attenuated PfSPZ, 4.5 × 105 PfSPZ, N = 13), or normal saline placebo at 8-week intervals, followed by exposure to mosquito bite controlled human malaria infection (CHMI). After CHMI, 3 of 25 volunteers from both PfSPZ-GA1 groups were sterilely protected, and the remaining 17 of 22 showed a patency ≥9 days (median patency in controls, 7 days; range, 7 to 9). All volunteers in the PfSPZ Vaccine control group developed parasitemia (median patency, 9 days; range, 7 to 12). Immunized groups exhibited a significant, dose-related increase in anti-Pf circumsporozoite protein (CSP) antibodies and Pf-specific interferon-γ (IFN-γ)-producing T cells. Although no definite conclusion can be drawn on the potential strength of protective efficacy of PfSPZ-GA1 Vaccine, the favorable safety profile and induced immune responses by PfSPZ-GA1 Vaccine warrant further clinical evaluation.