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.

Serologic Markers of Previous Malaria Exposure and Functional Antibodies Inhibiting Parasite Growth Are Associated With Parasite Kinetics Following a Plasmodium falciparum Controlled Human Infection.

BACKGROUND: We assessed the impact of exposure to Plasmodium falciparum on parasite kinetics, clinical symptoms, and functional immunity after controlled human malaria infection (CHMI) in 2 cohorts with different levels of previous malarial exposure. METHODS: Nine adult males with high (sero-high) and 10 with low (sero-low) previous exposure received 3200 P. falciparum sporozoites (PfSPZ) of PfSPZ Challenge by direct venous inoculation and were followed for 35 days for parasitemia by thick blood smear (TBS) and quantitative polymerase chain reaction. Endpoints were time to parasitemia, adverse events, and immune responses. RESULTS: Ten of 10 (100%) volunteers in the sero-low and 7 of 9 (77.8%) in the sero-high group developed parasitemia detected by TBS in the first 28 days (P = .125). The median time to parasitemia was significantly shorter in the sero-low group than the sero-high group (9 days [interquartile range {IQR} 7.5-11.0] vs 11.0 days [IQR 7.5-18.0], respectively; log-rank test, P = .005). Antibody recognition of sporozoites was significantly higher in the sero-high (median, 17.93 [IQR 12.95-24] arbitrary units [AU]) than the sero-low volunteers (median, 10.54 [IQR, 8.36-12.12] AU) (P = .006). Growth inhibitory activity was significantly higher in the sero-high (median, 21.8% [IQR, 8.15%-29.65%]) than in the sero-low group (median, 8.3% [IQR, 5.6%-10.23%]) (P = .025). CONCLUSIONS: CHMI was safe and well tolerated in this population. Individuals with serological evidence of higher malaria exposure were able to better control infection and had higher parasite growth inhibitory activity. CLINICAL TRIALS REGISTRATION: NCT03496454.

The XN-30 hematology analyzer for rapid sensitive detection of malaria: a diagnostic accuracy study.

BACKGROUND: Accurate and timely diagnosis of malaria is essential for disease management and surveillance. Thin and thick blood smear microscopy and malaria rapid diagnostic tests (RDTs) are standard malaria diagnostics, but both methods have limitations. The novel automated hematology analyzer XN-30 provides standard complete blood counts (CBC) as well as quantification of malaria parasitemia at the price of a CBC. This study assessed the accuracy of XN-30 for malaria detection in a controlled human malaria infection (CHMI) study and a phase 3 diagnostic accuracy study in Burkina Faso. METHODS: Sixteen healthy, malaria-naive CHMI participants were challenged with five Plasmodium falciparum-infected mosquitoes. Blood was sampled daily for XN-30, blood smear microscopy, and malaria qPCR. The accuracy study included patients aged > 3 months presenting with acute febrile illness. XN-30, microscopy, and rapid diagnostic tests (HRP-2/pLDH) were performed on site; qPCR was done in retrospect. The malaria reference standard was microscopy, and results were corrected for sub-microscopic cases. RESULTS: All CHMI participants became parasitemic by qPCR and XN-30 with a strong correlation for parasite density (R2 = 0.91; p < .0001). The XN-30 accurately monitored treatment and allowed detection of recrudescence. Out of 908 patients in the accuracy study, 241 had microscopic malaria (density 24-491,802 parasites/µL). The sensitivity and specificity of XN-30 compared to microscopy were 98.7% and 99.4% (PPV = 98.7%, NPV = 99.4%). Results were corrected for qPCR-confirmed sub-microscopic cases. Three microscopy-confirmed cases were not detected by XN-30. However, XN-30 detected 19/134 (14.2%) qPCR-confirmed cases missed by microscopy. Among qPCR-confirmed cases, XN-30 had a higher sensitivity (70.9% versus 66.4%; p = .0009) and similar specificity (99.6% versus 100%; p = .5) as microscopy. The accuracy of XN-30 for microscopic malaria was equal to or higher than HRP-2 and pLDH RDTs, respectively. CONCLUSIONS: The XN-30 is a novel, automated hematology analyzer that combines standard hemocytometry with rapid, objective, and robust malaria detection and quantification, ensuring prompt treatment of malaria and malaria anemia and follow-up of treatment response. TRIAL REGISTRATION: Both trials were registered on clinicaltrials.gov with respective identifiers NCT02836002 (CHMI trial) and NCT02669823 (diagnostic accuracy study).

Modest heterologous protection after Plasmodium falciparum sporozoite immunization: a double-blind randomized controlled clinical trial.

BACKGROUND: A highly efficacious vaccine is needed for malaria control and eradication. Immunization with Plasmodium falciparum NF54 parasites under chemoprophylaxis (chemoprophylaxis and sporozoite (CPS)-immunization) induces the most efficient long-lasting protection against a homologous parasite. However, parasite genetic diversity is a major hurdle for protection against heterologous strains. METHODS: We conducted a double-blind, randomized controlled trial in 39 healthy participants of NF54-CPS immunization by bites of 45 NF54-infected (n = 24 volunteers) or uninfected mosquitoes (placebo; n = 15 volunteers) against a controlled human malaria infection with the homologous NF54 or the genetically distinct NF135.C10 and NF166.C8 clones. Cellular and humoral immune assays were performed as well as genetic characterization of the parasite clones. RESULTS: NF54-CPS immunization induced complete protection in 5/5 volunteers against NF54 challenge infection at 14 weeks post-immunization, but sterilely protected only 2/10 and 1/9 volunteers against NF135.C10 and NF166.C8 challenge infection, respectively. Post-immunization plasma showed a significantly lower capacity to block heterologous parasite development in primary human hepatocytes compared to NF54. Whole genome sequencing showed that NF135.C10 and NF166.C8 have amino acid changes in multiple antigens targeted by CPS-induced antibodies. Volunteers protected against heterologous challenge were among the stronger immune responders to in vitro parasite stimulation. CONCLUSIONS: Although highly protective against homologous parasites, NF54-CPS-induced immunity is less effective against heterologous parasite clones both in vivo and in vitro. Our data indicate that whole sporozoite-based vaccine approaches require more potent immune responses for heterologous protection. TRIAL REGISTRATION: This trial is registered in clinicaltrials.gov, under identifier NCT02098590 .

Concentration of Plasmodium falciparum gametocytes in whole blood samples by magnetic cell sorting enhances parasite infection rates in mosquito feeding assays.

BACKGROUND: Mosquito-feeding assays are important tools to guide the development and support the evaluation of transmission-blocking interventions. These functional bioassays measure the sporogonic development of gametocytes in blood-fed mosquitoes. Measuring the infectivity of low gametocyte densities has become increasingly important in malaria elimination scenarios. This will pose challenges to the sensitivity and throughput of existing mosquito-feeding assay protocols. Here, different gametocyte concentration methods of blood samples were explored to optimize conditions for detection of positive mosquito infections. METHODS: Mature gametocytes of Plasmodium falciparum were diluted into whole blood samples of malaria-naïve volunteers. Standard centrifugation, Percoll gradient, magnetic cell sorting (MACS) enrichment were compared using starting blood volumes larger than the control (direct) feed. RESULTS: MACS gametocyte enrichment resulted in the highest infection intensity with statistically significant increases in mean oocyst density in 2 of 3 experiments (p = 0.0003; p ≤ 0.0001; p = 0.2348). The Percoll gradient and standard centrifugation procedures resulted in variable infectivity. A significant increase in the proportion of infected mosquitoes and oocyst density was found when larger volumes of gametocyte-infected blood were used with the MACS procedure. CONCLUSIONS: The current study demonstrates that concentration methods of P. falciparum gametocyte-infected whole blood samples can enhance transmission in mosquito-feeding assays. Gametocyte purification by MACS was the most efficient method, allowing the assessment of gametocyte infectivity in low-density gametocyte infections, as can be expected in natural or experimental conditions.

Semi-high-throughput detection of Plasmodium falciparum and Plasmodium vivax oocysts in mosquitoes using bead-beating followed by circumsporozoite ELISA and quantitative PCR.

BACKGROUND: The malaria infection status of mosquitoes is commonly determined by microscopic detection of oocysts on the dissected mosquito midgut. This method is labour-intensive, does not allow processing of large numbers of mosquitoes and can be challenging in terms of objective classification of oocysts. Here, a semi-high-throughput bead-beating ELISA method is proposed for detection of the circumsporozoite protein (CSP) followed by confirmation by quantitative PCR (qPCR). METHODS: Cultured Plasmodium falciparum gametocytes were offered to Anopheles stephensi mosquitoes and examined by microscopy. After bead-beating, mosquito homogenate was examined by CSP-ELISA and 18S qPCR. As negative controls, mosquitoes that were offered a heat-inactivated gametocyte blood meal were used. The CSP-ELISA/qPCR methodology was applied to high and low-intensity infections of cultured P. falciparum gametocytes. A similar methodology optimized for P. vivax was used on mosquitoes that were offered blood from Ethiopian donors who were naturally infected with P. vivax. RESULTS: There was considerable variation in CSP-ELISA signal and qPCR values in mosquitoes with low oocyst intensities. There was a strong agreement mosquito positivity by CSP-ELISA and by qPCR in mosquitoes that fed on cultured P. falciparum material (agreement 96.9%; kappa = 0.97) and naturally infected P. vivax parasite carriers [agreement 92.4% (kappa = 0.83)]. CONCLUSIONS: The proposed bead-beating CSP-ELISA/qPCR methodology considerably increases throughput for the detection of mosquito infection. qPCR remains necessary to confirm infections in mosquitoes with low CSP-ELISA signal. This methodology may prove particularly useful for studies where very low mosquito infection prevalence is expected and study sites where experience with oocyst detection is limited.

Diagnosis and treatment based on quantitative PCR after controlled human malaria infection.

BACKGROUND: Controlled human malaria infection (CHMI) has become well-established in the evaluation of drugs and vaccines. Anti-malarial treatment is usually initiated when thick blood smears are positive by microscopy. This study explores the effects of using the more sensitive qPCR as the primary diagnostic test. METHODS: 1691 diagnostic blood samples were analysed by microscopy and qPCR from 115 volunteers (55 malaria naïve and 60 having received chemoprophylaxis and sporozoite immunization) who were challenged by five mosquitoes infected with Plasmodium falciparum sporozoites of the NF54 strain. RESULTS: Retrospective analysis of different qPCR criteria for diagnosis and treatment, showed that once daily qPCR (threshold 100 parasites/ml) had 99 % sensitivity and 100 % specificity, and shortened the median prepatent period from 10.5 to 7.0 days after CHMI when compared to twice daily measurement of thick blood smears (threshold 4000 parasites/ml). This is expected to result in a 78 % decrease of adverse events before initiation of treatment in future studies. Trial outcome related to infection and protective efficacy remained unchanged. CONCLUSION: The use of qPCR as the primary diagnostic test in CHMI decreases symptoms as well as parasitaemia while obviating the need for twice daily follow-up. The implementation improves safety while reducing the clinical burden and costs without compromising the evaluation of protective efficacy.

Erratum to: Diagnosis and treatment based on quantitative PCR after controlled human malaria infection.

[This corrects the article DOI: 10.1186/s12936-016-1434-z.].

Impact of oral vancomycin on gut microbiota, bile acid metabolism, and insulin sensitivity.

BACKGROUND & AIMS: Obesity has been associated with changes in the composition and function of the intestinal microbiota. Modulation of the microbiota by antibiotics also alters bile acid and glucose metabolism in mice. Hence, we hypothesized that short term administration of oral antibiotics in humans would affect fecal microbiota composition and subsequently bile acid and glucose metabolism. METHODS: In this single blinded randomized controlled trial, 20 male obese subjects with metabolic syndrome were randomized to 7 days of amoxicillin 500 mg t.i.d. or 7 days of vancomycin 500 mg t.i.d. At baseline and after 1 week of therapy, fecal microbiota composition (Human Intestinal Tract Chip phylogenetic microarray), fecal and plasma bile acid concentrations as well as insulin sensitivity (hyperinsulinemic euglycemic clamp using [6,6-(2)H2]-glucose tracer) were measured. RESULTS: Vancomycin reduced fecal microbial diversity with a decrease of gram-positive bacteria (mainly Firmicutes) and a compensatory increase in gram-negative bacteria (mainly Proteobacteria). Concomitantly, vancomycin decreased fecal secondary bile acids with a simultaneous postprandial increase in primary bile acids in plasma (p<0.05). Moreover, changes in fecal bile acid concentrations were predominantly associated with altered Firmicutes. Finally, administration of vancomycin decreased peripheral insulin sensitivity (p<0.05). Amoxicillin did not affect any of these parameters. CONCLUSIONS: Oral administration of vancomycin significantly impacts host physiology by decreasing intestinal microbiota diversity, bile acid dehydroxylation and peripheral insulin sensitivity in subjects with metabolic syndrome. These data show that intestinal microbiota, particularly of the Firmicutes phylum contributes to bile acid and glucose metabolism in humans. This trial is registered at the Dutch Trial Register (NTR2566).

Controlled human malaria infections by mosquito bites induce more severe clinical symptoms than asexual blood-stage challenge infections.

BACKGROUND: Fever and inflammation are a hallmark of clinical Plasmodium falciparum (Pf) malaria induced by circulating asexual parasites. Although clinical manifestations of inflammation are associated with parasite density, this relationship is influenced by a complex network of immune-modulating factors of both human and parasite origin. METHODS: In the Controlled Human Malaria infection (CHMI) model, we compared clinical inflammation in healthy malaria-naïve volunteers infected by either Pf-infected mosquito bites (MB, n=12) or intravenous administration of Pf-infected red blood cells (BS, n=12). FINDINGS: All volunteers developed patent parasitaemia, but both the incidence and duration of severe adverse events were significantly higher after MB infection. Similarly, clinical laboratory markers of inflammation were significantly increased in the MB-group, as well as serum pro-inflammatory cytokine concentrations including IFN-γ, IL-6, MCP1 and IL-8. Parasite load, as reflected by maximum parasite density and area under the curve, was similar, but median duration of parasitaemia until treatment was longer in the BS-group compared to the MB-group (8 days [range 8 - 8 days] versus 5·5 days [range 3·5 - 12·5 days]). The in vitro response of subsets of peripheral blood mononuclear cells showed attenuated Pf-specific IFNγ production by γδ T-cells in the BS-arm. INTERPRETATION: In conclusion, irrespective the parasite load, Pf-infections by MB induce stronger signs and symptoms of inflammation compared to CHMI by BS infection. The pathophysiological basis remains speculative but may relate to induced immune tolerance. FUNDING: The trial was supported by PATH's Malaria Vaccine Initiative; the current analyses were supported by the AMMODO Science Award 2019 (TB).

Functional changes in hemostasis during asexual and sexual parasitemia in a controlled human malaria infection.

Decreased platelet count is an early phenomenon in asexual Plasmodium falciparum parasitemia, but its association with acute or long-term functional changes in platelets and coagulation is unknown. Moreover, the impact of gametocytemia on platelets and coagulation remains unclear. We investigated the changes in platelet number and function during early asexual parasitemia, gametocytemia and convalescence in 16 individuals participating in a controlled human malaria infection study, and studied its relationship with changes in total and active von Willebrand factor levels (VWF) and the coagulation system. Platelet activation and reactivity were determined by flow cytometry, and the coagulation system was assessed using different representative assays including antigen assays, activity assays and global functional assays. Platelet count was decreased during asexual blood stage infection but normalized during gametocytemia. Platelet P-selectin expression was slightly increased during asexual parasitemia, gametocytemia and at day 64. In contrast, platelet reactivity to different agonists remained unchanged, except a marked decrease in reactivity to low dose collagen-related peptide-XL. Thrombin generation and antigen assays did not show a clear activation of the coagulation during asexual parasitemia, whereas total and active VWF levels were markedly increased. During gametocytemia and on day 64, the endogenous thrombin potential, thrombin peak and velocity index were increased and prothrombin conversion and plasma prothrombin levels were decreased. We conclude that the decreased platelet count during asexual parasitemia is associated with increased active VWF levels (i.e. endothelial activation), but not platelet hyperreactivity or hypercoagulability, and that the increased platelet clearance in asexual parasitemia could cause spontaneous VWF-platelet complexes formation.

Biomarkers of cellular aging during a controlled human malaria infection.

Cellular aging is difficult to study in individuals with natural infection, given the diversity of symptom duration and clinical presentation, and the high interference of aging-related processes with host and environmental factors. To address this challenge, we took advantage of the controlled human malaria infection (CHMI) model. This approach allowed us to characterize the relationship among cellular aging markers prior, during and post malaria pathophysiology in humans, controlling for infection dose, individual heterogeneity, previous exposure and co-infections. We demonstrate that already low levels of Plasmodium falciparum impact cellular aging by inducing high levels of inflammation and redox-imbalance; and that cellular senescence reversed after treatment and parasite clearance. This study provides insights into the complex relationship of telomere length, cellular senescence, telomerase expression and aging-related processes during a single malaria infection.

An open-label phase 1/2a trial of a genetically modified rodent malaria parasite for immunization against Plasmodium falciparum malaria.

For some diseases, successful vaccines have been developed using a nonpathogenic counterpart of the causative microorganism of choice. The nonpathogenicity of the rodent Plasmodium berghei (Pb) parasite in humans prompted us to evaluate its potential as a platform for vaccination against human infection by Plasmodium falciparum (Pf), a causative agent of malaria. We hypothesized that the genetic insertion of a leading protein target for clinical development of a malaria vaccine, Pf circumsporozoite protein (CSP), in its natural pre-erythrocytic environment, would enhance Pb's capacity to induce protective immunity against Pf infection. Hence, we recently generated a transgenic Pb sporozoite immunization platform expressing PfCSP (PbVac), and we now report the clinical evaluation of its biological activity against controlled human malaria infection (CHMI). This first-in-human trial shows that PbVac is safe and well tolerated, when administered by a total of ~300 PbVac-infected mosquitoes per volunteer. Although protective efficacy evaluated by CHMI showed no sterile protection at the tested dose, significant delays in patency (2.2 days, P = 0.03) and decreased parasite density were observed after immunization, corresponding to an estimated 95% reduction in Pf liver parasite burden (confidence interval, 56 to 99%; P = 0.010). PbVac elicits dose-dependent cross-species cellular immune responses and functional PfCSP-dependent antibody responses that efficiently block Pf sporozoite invasion of liver cells in vitro. This study demonstrates that PbVac immunization elicits a marked biological effect, inhibiting a subsequent infection by the human Pf parasite, and establishes the clinical validation of a new paradigm in malaria vaccination.

A controlled human malaria infection model enabling evaluation of transmission-blocking interventions.

BACKGROUND: Drugs and vaccines that can interrupt the transmission of Plasmodium falciparum will be important for malaria control and elimination. However, models for early clinical evaluation of candidate transmission-blocking interventions are currently unavailable. Here, we describe a new model for evaluating malaria transmission from humans to Anopheles mosquitoes using controlled human malaria infection (CHMI). METHODS: Seventeen healthy malaria-naive volunteers underwent CHMI by intravenous inoculation of P. falciparum-infected erythrocytes to initiate blood-stage infection. Seven to eight days after inoculation, participants received piperaquine (480 mg) to attenuate asexual parasite replication while allowing gametocytes to develop and mature. Primary end points were development of gametocytemia, the transmissibility of gametocytes from humans to mosquitoes, and the safety and tolerability of the CHMI transmission model. To investigate in vivo gametocytocidal drug activity in this model, participants were either given an experimental antimalarial, artefenomel (500 mg), or a known gametocytocidal drug, primaquine (15 mg), or remained untreated during the period of gametocyte carriage. RESULTS: Male and female gametocytes were detected in all participants, and transmission to mosquitoes was achieved from 8 of 11 (73%) participants evaluated. Compared with results in untreated controls (n = 7), primaquine (15 mg, n = 5) significantly reduced gametocyte burden (P = 0.01), while artefenomel (500 mg, n = 4) had no effect. Adverse events (AEs) were mostly mild or moderate. Three AEs were assessed as severe - fatigue, elevated alanine aminotransferase, and elevated aspartate aminotransferase - and were attributed to malaria infection. Transaminase elevations were transient, asymptomatic, and resolved without intervention. CONCLUSION: We report the safe and reproducible induction of P. falciparum gametocytes in healthy malaria-naive volunteers at densities infectious to mosquitoes, thereby demonstrating the potential for evaluating transmission-blocking interventions in this model. TRIAL REGISTRATION: ClinicalTrials.gov NCT02431637 and NCT02431650. FUNDING: Bill & Melinda Gates Foundation.

Antibody Responses to Antigenic Targets of Recent Exposure Are Associated With Low-Density Parasitemia in Controlled Human Plasmodium falciparum Infections.

The majority of malaria infections in low transmission settings remain undetectable by conventional diagnostics. A powerful model to identify antibody responses that allow accurate detection of recent exposure to low-density infections is controlled human malaria infection (CHMI) studies in which healthy volunteers are infected with the Plasmodium parasite. We aimed to evaluate antibody responses in malaria-naïve volunteers exposed to a single CHMI using a custom-made protein microarray. All participants developed a blood-stage infection with peak parasite densities up to 100 parasites/µl in the majority of participants (50/54), while the remaining four participants had peak densities between 100 and 200 parasites/µl. There was a strong correlation between parasite density and antibody responses associated with the most reactive blood-stage targets 1 month after CHMI (Etramp 5, GLURP-R2, MSP4 and MSP1-19; Spearman's ρ = 0.82, p < 0.001). Most volunteers developed antibodies against a potential marker of recent exposure: Etramp 5 (37/45, 82%). Our findings justify validation in endemic populations to define a minimum set of antigens needed to detect exposure to natural low-density infections.

Pre-clinical evaluation of a P. berghei-based whole-sporozoite malaria vaccine candidate.

Whole-sporozoite vaccination/immunization induces high levels of protective immunity in both rodent models of malaria and in humans. Recently, we generated a transgenic line of the rodent malaria parasite P. berghei (Pb) that expresses the P. falciparum (Pf) circumsporozoite protein (PfCS), and showed that this parasite line (PbVac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human Pf parasite. Here, we analyzed PbVac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of PbVac parasites was generated through a process of cyclic propagation and clonal expansion in mice and mosquitoes and was genetically characterized. A highly sensitive qRT-PCR-based method was established that enables PbVac parasite detection and quantification at low parasite densities in vivo. This method was employed in a biodistribution study in a rabbit model, revealing that the parasite is only present at the site of administration and in the liver up to 48 h post infection and is no longer detectable at any site 10 days after administration. An extensive toxicology investigation carried out in rabbits further showed the absence of PbVac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of PbVac were completely eliminated by Malarone® treatment. Collectively, our pre-clinical safety assessment demonstrates that PbVac possesses all characteristics necessary to advance into clinical evaluation.

A Plasmodium berghei sporozoite-based vaccination platform against human malaria.

There is a pressing need for safe and highly effective Plasmodium falciparum (Pf) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei (Pb) parasites as cross-species immunizing agents and as platforms for expression and delivery of PfCS (PbVac). We show that both wild-type Pb and PbVac sporozoites unabatedly infect and develop in human hepatocytes while unable to establish an infection in human red blood cells. In a rabbit model, similarly susceptible to Pb hepatic but not blood infection, we show that PbVac elicits cross-species cellular immune responses, as well as PfCS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, PbVac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.

A randomized feasibility trial comparing four antimalarial drug regimens to induce Plasmodium falciparum gametocytemia in the controlled human malaria infection model.

Background: Malaria elimination strategies require a thorough understanding of parasite transmission from human to mosquito. A clinical model to induce gametocytes to understand their dynamics and evaluate transmission-blocking interventions (TBI) is currently unavailable. Here, we explore the use of the well-established Controlled Human Malaria Infection model (CHMI) to induce gametocyte carriage with different antimalarial drug regimens. Methods: In a single centre, open-label randomised trial, healthy malaria-naive participants (aged 18­35 years) were infected with Plasmodium falciparum by bites of infected Anopheles mosquitoes. Participants were randomly allocated to four different treatment arms (n = 4 per arm) comprising low-dose (LD) piperaquine (PIP) or sulfadoxine-pyrimethamine (SP), followed by a curative regimen upon recrudescence. Male and female gametocyte densities were determined by molecular assays. Results: Mature gametocytes were observed in all participants (16/16, 100%). Gametocytes appeared 8.5­12 days after the first detection of asexual parasites. Peak gametocyte densities and gametocyte burden was highest in the LD-PIP/SP arm, and associated with the preceding asexual parasite biomass (p=0.026). Male gametocytes had a mean estimated circulation time of 2.7 days (95% CI 1.5­3.9) compared to 5.1 days (95% CI 4.1­6.1) for female gametocytes. Exploratory mosquito feeding assays showed successful sporadic mosquito infections. There were no serious adverse events or significant differences in the occurrence and severity of adverse events between study arms (p=0.49 and p=0.28). Conclusions: The early appearance of gametocytes indicates gametocyte commitment during the first wave of asexual parasites emerging from the liver. Treatment by LD-PIP followed by a curative SP regimen, results in the highest gametocyte densities and the largest number of gametocyte-positive days. This model can be used to evaluate the effect of drugs and vaccines on gametocyte dynamics, and lays the foundation for fulfilling the critical unmet need to evaluate transmission-blocking interventions against falciparum malaria for downstream selection and clinical development. Funding: Funded by PATH Malaria Vaccine Initiative (MVI). Clinical trial number: NCT02836002.

The parasite that causes malaria, named Plasmodium falciparum, has a life cycle that involves both humans and mosquitoes. Starting in the saliva of female Anopheles mosquitoes, it enters a person's bloodstream when the insects feed. It then moves to the person's liver, where it infects liver cells and matures into a stage known as schizonts. The schizonts then divide to form thousands of so-called merozoites, which burst out of the liver cells and into the bloodstream. The merozoites infect red blood cells, producing more schizonts and yet more merozoites, which continue the infection. To complete its life cycle, the parasite must return to a mosquito. Some of the parasites in the person's blood transform into male and female cells called gametocytes that are taken up by a mosquito when it feeds on that person. Inside the mosquito, male and female parasites reproduce to create the next generation of parasites. The new parasites then move to the mosquito's salivary glands, ready to begin another infection. Stopping the parasite being transmitted from humans to mosquitoes will stop the spread of malaria in the population. Yet it has proven difficult to study this part of the life cycle from natural infections. Here, Reuling et al. report a new method for generating gametocytes in human volunteers that will enable closer study of the biology of malaria transmission. The method is developed using the Controlled Human Malaria Infection (CHMI) model. Healthy volunteers without a history of malaria are bitten by mosquitoes infected with malaria parasites. Shortly afterwards, the volunteers are given a drug treatment to control and reduce their symptoms. The gametocytes form during this phase of the infection. At the end of the experiment, all the volunteers receive a final treatment that completely cures the infection. Reuling et al. recruited 16 volunteers and assigned them to four groups at random. Each group received a different drug regime. Roughly a week after the mosquito bites, all participants showed malaria parasites in their blood, and between 8.5 and 12 days later, mature gametocytes started to appear. This early appearance suggests that the parasites start to transform into gametocytes when they first emerge from the liver. The experiment also revealed that female gametocytes stay in the blood for a longer period than their male counterparts. These results are proof of principle for a new way to investigate malaria infection. The new model provides a controlled method for studying P. falciparum gametocytes in people. In the future, it could help to test the impact of drugs and vaccines on gametocytes. Understanding more about these parasites' biology could lead to treatments that block malaria transmission.

Humoral protection against mosquito bite-transmitted Plasmodium falciparum infection in humanized mice.

A malaria vaccine that prevents infection will be an important new tool in continued efforts of malaria elimination, and such vaccines are under intense development for the major human malaria parasite Plasmodium falciparum (Pf). Antibodies elicited by vaccines can block the initial phases of parasite infection when sporozoites are deposited into the skin by mosquito bite and then target the liver for further development. However, there are currently no standardized in vivo preclinical models that can measure the inhibitory activity of antibody specificities against Pf sporozoite infection via mosquito bite. Here, we use human liver-chimeric mice as a challenge model to assess prevention of natural Pf sporozoite infection by antibodies. We demonstrate that these mice are consistently infected with Pf by mosquito bite and that this challenge can be combined with passive transfer of either monoclonal antibodies or polyclonal human IgG from immune serum to measure antibody-mediated blocking of parasite infection using bioluminescent imaging. This methodology is useful to down-select functional antibodies and to investigate mechanisms or immune correlates of protection in clinical trials, thereby informing rational vaccine optimization.