Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Human Antibodies that Slow Erythrocyte Invasion Potentiate Malaria-Neutralizing Antibodies.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the leading target for next-generation vaccines against the disease-causing blood-stage of malaria. However, little is known about how human antibodies confer functional immunity against this antigen. We isolated a panel of human monoclonal antibodies (mAbs) against PfRH5 from peripheral blood B cells from vaccinees in the first clinical trial of a PfRH5-based vaccine. We identified a subset of mAbs with neutralizing activity that bind to three distinct sites and another subset of mAbs that are non-functional, or even antagonistic to neutralizing antibodies. We also identify the epitope of a novel group of non-neutralizing antibodies that significantly reduce the speed of red blood cell invasion by the merozoite, thereby potentiating the effect of all neutralizing PfRH5 antibodies as well as synergizing with antibodies targeting other malaria invasion proteins. Our results provide a roadmap for structure-guided vaccine development to maximize antibody efficacy against blood-stage malaria.

Characterizing the Blood-Stage Antimalarial Activity of Tafenoquine in Healthy Volunteers Experimentally Infected With Plasmodium falciparum.

BACKGROUND: The long-acting 8-aminoquinoline tafenoquine may be a good candidate for mass drug administration if it exhibits sufficient blood-stage antimalarial activity at doses low enough to be tolerated by glucose 6-phosphate dehydrogenase (G6PD)-deficient individuals. METHODS: Healthy adults with normal levels of G6PD were inoculated with Plasmodium falciparum 3D7-infected erythrocytes on day 0. Different single oral doses of tafenoquine were administered on day 8. Parasitemia and concentrations of tafenoquine and the 5,6-orthoquinone metabolite in plasma/whole blood/urine were measured and standard safety assessments performed. Curative artemether-lumefantrine therapy was administered if parasite regrowth occurred, or on day 48 ± 2. Outcomes were parasite clearance kinetics, pharmacokinetic and pharmacokinetic/pharmacodynamic (PK/PD) parameters from modelling, and dose simulations in a theoretical endemic population. RESULTS: Twelve participants were inoculated and administered 200 mg (n = 3), 300 mg (n = 4), 400 mg (n = 2), or 600 mg (n = 3) tafenoquine. The parasite clearance half-life with 400 mg or 600 mg (5.4 hours and 4.2 hours, respectively) was faster than with 200 mg or 300 mg (11.8 hours and 9.6 hours, respectively). Parasite regrowth occurred after dosing with 200 mg (3/3 participants) and 300 mg (3/4 participants) but not after 400 mg or 600 mg. Simulations using the PK/PD model predicted that 460 mg and 540 mg would clear parasitaemia by a factor of 106 and 109, respectively, in a 60-kg adult. CONCLUSIONS: Although a single dose of tafenoquine exhibits potent P. falciparum blood-stage antimalarial activity, the estimated doses to effectively clear asexual parasitemia will require prior screening to exclude G6PD deficiency. Clinical Trials Registration. Australian and New Zealand Clinical Trials Registry (ACTRN12620000995976).

Assessment of precision in growth inhibition assay (GIA) using human anti-PfRH5 antibodies.

BACKGROUND: For blood-stage malaria vaccine development, the in vitro growth inhibition assay (GIA) has been widely used to evaluate functionality of vaccine-induced antibodies (Ab), and Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage antigen. However, precision, also called "error of assay (EoA)", in GIA readouts and the source of EoA has not been evaluated systematically. METHODS: In the Main GIA experiment, 4 different cultures of P. falciparum 3D7 parasites were prepared with red blood cells (RBC) collected from 4 different donors. For each culture, 7 different anti-RH5 Ab (either monoclonal or polyclonal Ab) were tested by GIA at two concentrations on three different days (168 data points). To evaluate sources of EoA in % inhibition in GIA (%GIA), a linear model fit was conducted including donor (source of RBC) and day of GIA as independent variables. In addition, 180 human anti-RH5 polyclonal Ab were tested in a Clinical GIA experiment, where each Ab was tested at multiple concentrations in at least 3 independent GIAs using different RBCs (5,093 data points). The standard deviation (sd) in %GIA and in GIA50 (Ab concentration that gave 50%GIA) readouts, and impact of repeat assays on 95% confidence interval (95%CI) of these readouts was estimated. RESULTS: The Main GIA experiment revealed that the RBC donor effect was much larger than the day effect, and an obvious donor effect was also observed in the Clinical GIA experiment. Both %GIA and log-transformed GIA50 data reasonably fit a constant sd model, and sd of %GIA and log-transformed GIA50 measurements were calculated as 7.54 and 0.206, respectively. Taking the average of three repeat assays (using three different RBCs) reduces the 95%CI width in %GIA or in GIA50 measurements by ~ half compared to a single assay. CONCLUSIONS: The RBC donor effect (donor-to-donor variance on the same day) in GIA was much bigger than the day effect (day-to-day variance using the same donor's RBC) at least for the RH5 Ab evaluated in this study; thus, future GIA studies should consider the donor effect. In addition, the 95%CI for %GIA and GIA50 shown here help when comparing GIA results from different samples/groups/studies; therefore, this study supports future malaria blood-stage vaccine development.

Patient variability in the blood-stage dynamics of Plasmodium falciparum captured by clustering historical data.

BACKGROUND: Mathematical models provide an understanding of the dynamics of a Plasmodium falciparum blood-stage infection (within-host models), and can predict the impact of control strategies that affect the blood-stage of malaria. However, the dynamics of P. falciparum blood-stage infections are highly variable between individuals. Within-host models use different techniques to capture this inter-individual variation. This struggle may be unnecessary because patients can be clustered according to similar key within-host dynamics. This study aimed to identify clusters of patients with similar parasitaemia profiles so that future mathematical models can include an improved understanding of within-host variation. METHODS: Patients' parasitaemia data were analyzed to identify (i) clusters of patients (from 35 patients) that have a similar overall parasitaemia profile and (ii) clusters of patients (from 100 patients) that have a similar first wave of parasitaemia. For each cluster analysis, patients were clustered based on key features which previous models used to summarize parasitaemia dynamics. The clustering analyses were performed using a finite mixture model. The centroid values of the clusters were used to parameterize two established within-host models to generate parasitaemia profiles. These profiles (that used the novel centroid parameterization) were compared with profiles that used individual-specific parameterization (as in the original models), as well as profiles that ignored individual variation (using overall means for parameterization). RESULTS: To capture the variation of within-host dynamics, when studying the overall parasitaemia profile, two clusters efficiently grouped patients based on their infection length and the height of the first parasitaemia peak. When studying the first wave of parasitaemia, five clusters efficiently grouped patients based on the height of the peak and the speed of the clearance following the peak of parasitaemia. The clusters were based on features that summarize the strength of patient innate and adaptive immune responses. Parameterizing previous within host-models based on cluster centroid values accurately predict individual patient parasitaemia profiles. CONCLUSION: This study confirms that patients have personalized immune responses, which explains the variation of parasitaemia dynamics. Clustering can guide the optimal inclusion of within-host variation in future studies, and inform the design and parameterization of population-based models.

Antibody Combinations Targeting the Essential Antigens CyRPA, RH5, and MSP-119 Potently Neutralize Plasmodium falciparum Clinical Isolates From India and Africa.

BACKGROUND: Targeting multiple key antigens that mediate distinct Plasmodium falciparum erythrocyte invasion pathways is an attractive approach for the development of blood-stage malaria vaccines. However, the challenge is to identify antigen cocktails that elicit potent strain-transcending parasite-neutralizing antibodies efficacious at low immunoglobulin G concentrations feasible to achieve through vaccination. Previous reports have screened inhibitory antibodies primarily against well adapted laboratory parasite clones. However, validation of the parasite-neutralizing efficacy against clinical isolates with minimal in vitro cultivation is equally significant to better ascertain their prospective in vivo potency. METHODS: We evaluated the parasite-neutralizing activity of different antibodies individually and in combinations against laboratory adapted clones and clinical isolates. Clinical isolates were collected from Central India and Mozambique, Africa, and characterized for their invasion properties and genetic diversity of invasion ligands. RESULTS: In our portfolio, we evaluated 25 triple antibody combinations and identified the MSP-Fu+CyRPA+RH5 antibody combination to elicit maximal parasite neutralization against P. falciparum clinical isolates with variable properties that underwent minimal in vitro cultivation. CONCLUSIONS: The MSP-Fu+CyRPA+RH5 combination exhibited highly robust parasite neutralization against P. falciparum clones and clinical isolates, thus substantiating them as promising candidate antigens and establishing a proof of principle for the development of a combinatorial P. falciparum blood-stage malaria vaccine.

Semimechanistic Pharmacokinetic and Pharmacodynamic Modeling of Piperaquine in a Volunteer Infection Study with Plasmodium falciparum Blood-Stage Malaria.

Dihydroartemisinin-piperaquine is a recommended first-line artemisinin combination therapy for Plasmodium falciparum malaria. Piperaquine is also under consideration for other antimalarial combination therapies. The aim of this study was to develop a pharmacokinetic-pharmacodynamic model that might be useful when optimizing the use of piperaquine in new antimalarial combination therapies. The pharmacokinetic-pharmacodynamic model was developed using data from a previously reported dose-ranging study where 24 healthy volunteers were inoculated with 1,800 blood-stage Plasmodium falciparum parasites. All volunteers received a single oral dose of piperaquine (960 mg, 640 mg, or 480 mg) on day 7 or day 8 after parasite inoculation in separate cohorts. Parasite densities were measured by quantitative PCR (qPCR), and piperaquine levels were measured in plasma samples. We used nonlinear mixed-effect modeling to characterize the pharmacokinetic properties of piperaquine and the parasite dynamics associated with piperaquine exposure. The pharmacokinetics of piperaquine was described by a three-compartment disposition model. A semimechanistic parasite dynamics model was developed to explain the maturation of parasites, sequestration of mature parasites, synchronicity of infections, and multiplication of parasites, as seen in natural clinical infections with P. falciparum malaria. Piperaquine-associated parasite killing was estimated using a maximum effect (Emax) function. Treatment simulations (i.e., 3-day oral dosing of dihydroartemisinin-piperaquine) indicated that to be able to combat multidrug-resistant infections, an ideal additional drug in a new antimalarial triple-combination therapy should have a parasite reduction ratio of ≥102 per life cycle (38.8 h) with a duration of action of ≥2 weeks. The semimechanistic pharmacokinetic-pharmacodynamic model described here offers the potential to be a valuable tool for assessing and optimizing current and new antimalarial drug combination therapies containing piperaquine and the impact of these therapies on killing multidrug-resistant infections. (This study has been registered in the Australian and New Zealand Clinical Trials Registry under no. ANZCTRN12613000565741.).

The PHIST protein GEXP02 targets the host cytoskeleton during sexual development of Plasmodium falciparum.

A hallmark of the biology of Plasmodium falciparum blood stage parasites is their extensive host cell remodelling, facilitated by parasite proteins that are exported into the erythrocyte. Although this area has received extensive attention, only a few exported parasite proteins have been analysed in detail, and much of this remodelling process remains unknown, particularly for gametocyte development. Recent advances to induce high rates of sexual commitment enable the production of large numbers of gametocytes. We used this approach to study the Plasmodium helical interspersed subtelomeric (PHIST) protein GEXP02, which is expressed during sexual development. We show by immunofluorescence that GEXP02 is exported to the gametocyte-infected host cell periphery. Co-immunoprecipitation revealed potential interactions between GEXP02 and components of the erythrocyte cytoskeleton as well as other exported parasite proteins. This indicates that GEXP02 targets the erythrocyte cytoskeleton and is likely involved in its remodelling. GEXP02 knock-out parasites show no obvious phenotype during gametocyte maturation, transmission through mosquitoes, and hepatocyte infection, suggesting auxiliary or redundant functions for this protein. In summary, we performed a detailed cellular and biochemical analysis of a sexual stage-specific exported parasite protein using a novel experimental approach that is broadly applicable to study the biology of P. falciparum gametocytes.

Haematological response in experimental human Plasmodium falciparum and Plasmodium vivax malaria.

BACKGROUND: Malaria-associated anaemia, arising from symptomatic, asymptomatic and submicroscopic infections, is a significant cause of morbidity worldwide. Induced blood stage malaria volunteer infection studies (IBSM-VIS) provide a unique opportunity to evaluate the haematological response to early Plasmodium falciparum and Plasmodium vivax infection. METHODS: This study was an analysis of the haemoglobin, red cell counts, and parasitaemia data from 315 participants enrolled in IBSM-VIS between 2012 and 2019, including 269 participants inoculated with the 3D7 strain of P. falciparum (Pf3D7), 15 with an artemisinin-resistant P. falciparum strain (PfK13) and 46 with P. vivax. Factors associated with the fractional fall in haemoglobin (Hb-FF) were evaluated, and the malaria-attributable erythrocyte loss after accounting for phlebotomy-related losses was estimated. The relative contribution of parasitized erythrocytes to the malaria-attributable erythrocyte loss was also estimated. RESULTS: The median peak parasitaemia prior to treatment was 10,277 parasites/ml (IQR 3566-27,815), 71,427 parasites/ml [IQR 33,236-180,213], and 34,840 parasites/ml (IQR 13,302-77,064) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. The median Hb-FF was 10.3% (IQR 7.8-13.3), 14.8% (IQR 11.8-15.9) and 11.7% (IQR 8.9-14.5) in those inoculated with Pf3D7, PfK13 and P. vivax, respectively, with the haemoglobin nadir occurring a median 12 (IQR 5-21), 15 (IQR 7-22), and 8 (IQR 7-15) days following inoculation. In participants inoculated with P. falciparum, recrudescence was associated with a greater Hb-FF, while in those with P. vivax, the Hb-FF was associated with a higher pre-treatment parasitaemia and later day of anti-malarial treatment. After accounting for phlebotomy-related blood losses, the estimated Hb-FF was 4.1% (IQR 3.1-5.3), 7.2% (IQR 5.8-7.8), and 4.9% (IQR 3.7-6.1) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. Parasitized erythrocytes were estimated to account for 0.015% (IQR 0.006-0.06), 0.128% (IQR 0.068-0.616) and 0.022% (IQR 0.008-0.082) of the malaria-attributable erythrocyte loss in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. CONCLUSION: Early experimental P. falciparum and P. vivax infection resulted in a small but significant fall in haemoglobin despite parasitaemia only just at the level of microscopic detection. Loss of parasitized erythrocytes accounted for < 0.2% of the total malaria-attributable haemoglobin loss.

Development and evaluation of a new Plasmodium falciparum 3D7 blood stage malaria cell bank for use in malaria volunteer infection studies.

BACKGROUND: New anti-malarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating anti-malarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. This study aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated. METHODS: The 3D7-V2 MCB was expanded in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. RESULTS: The in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and < 0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable P. falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 h (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI 18.5-64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI 8.5-15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI 3.61-4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI 4.16-4.47]) hours for 3D7-V1. A total of 59 adverse events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study. CONCLUSION: The safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials registry numbers: P3487 (3D7-V1): ACTRN12619001085167. P3491 (3D7-MBE-008): ACTRN12619001079134.

An Experimental Human Blood-Stage Model for Studying Plasmodium malariae Infection.

BACKGROUND: Plasmodium malariae is considered a minor malaria parasite, although its global disease burden is underappreciated. The aim of this study was to develop an induced blood-stage malaria (IBSM) model of P. malariae to study parasite biology, diagnostic assays, and treatment. METHODS: This clinical trial involved 2 healthy subjects who were intravenously inoculated with cryopreserved P. malariae-infected erythrocytes. Subjects were treated with artemether-lumefantrine after development of clinical symptoms. Prior to antimalarial therapy, mosquito-feeding assays were performed to investigate transmission, and blood samples were collected for rapid diagnostic testing and parasite transcription profiling. Serial blood samples were collected for biomarker analysis. RESULTS: Both subjects experienced symptoms and signs typical of early malaria. Parasitemia was detected 7 days after inoculation, and parasite concentrations increased until antimalarial treatment was initiated 25 and 21 days after inoculation for subjects 1 and 2 respectively (peak parasitemia levels, 174 182 and 50 291 parasites/mL, respectively). The parasite clearance half-life following artemether-lumefantrine treatment was 6.7 hours. Mosquito transmission was observed for 1 subject, while in vivo parasite transcription and biomarkers were successfully profiled. CONCLUSIONS: An IBSM model of P. malariae has been successfully developed and may be used to study the biology of, diagnostic testing for, and treatment of this neglected malaria species. CLINICAL TRIALS REGISTRATION: ACTRN12617000048381.

Growth Rate of Plasmodium falciparum: Analysis of Parasite Growth Data From Malaria Volunteer Infection Studies.

BACKGROUND: Growth rate of malaria parasites in the blood of infected subjects is an important measure of efficacy of drugs and vaccines. METHODS: We used log-linear and sine-wave models to estimate the parasite growth rate of the 3D7 strain of Plasmodium falciparum using data from 177 subjects from 14 induced blood stage malaria (IBSM) studies conducted at QIMR Berghofer. We estimated parasite multiplication rate per 48 hours (PMR48), PMR per life-cycle (PMRLC), and parasite life-cycle duration. We compared these parameters to those from studies conducted elsewhere with infections induced by IBSM (n = 66), sporozoites via mosquito bite (n = 336), or injection (n = 51). RESULTS: The parasite growth rate of 3D7 in QIMR Berghofer studies was 0.75/day (95% confidence interval [CI], .73-.77/day), PMR48 was 31.9 (95% CI, 28.7-35.4), PMRLC was 16.4 (95% CI, 15.1-17.8), and parasite life-cycle was 38.8 hours (95% CI, 38.3-39.2 hours). These parameters were similar to estimates from IBSM studies elsewhere (0.71/day, 95% CI, .67-.75/day; PMR48 26.6, 95% CI, 22.2-31.8) but significantly higher (P < .001) than in sporozoite studies (0.47/day, 95% CI, .43-.50/day; PMR48 8.6, 95% CI, 7.3-10.1). CONCLUSIONS: Parasite growth rates were similar across different IBSM studies and higher than infections induced by sporozoite.

Improving the immunogenicity and protective efficacy of a whole-killed malaria blood-stage vaccine by chloroquine.

A whole-killed malaria blood-stage vaccine (WKV) is promising in reducing the morbidity and mortality of malaria patients, but its efficacy needs to be improved. We found that the antimalarial drug chloroquine could augment the protective efficacy of the WKV of Plasmodium yoelii. The direct antimalarial effect of chloroquine on parasites during immunization could be excluded, as the administration of chloroquine or chloroquine plus alum every two weeks had a slight effect on parasitemia, and an immunization with NP-KLH (4-hydroxy-3-nitrophenylacetyl Keyhole Limpet Hemocyanin) plus chloroquine could significantly promote the generation of NP-specific antibodies. Additionally, alum was required for chloroquine to augment the immunogenicity of the pRBC lysate. Chloroquine did not promote the parasite-specific CD4+ T-cell responses, but significantly enhanced the WKV-induced germinal centre B cell reactions, class-switch recombination and secretion of functionally protective antibodies to plasmodium. The elevated parasite-specific antibodies were demonstrated to largely contribute to the chloroquine-enhanced protective immunity. Thus, we report that chloroquine could be used as an adjuvant to enhance the protective immunity of WKVs through promoting humoral responses.

Vaccination accelerates hepatic erythroblastosis induced by blood-stage malaria.

BACKGROUND: Vaccination induces survival of otherwise lethal blood-stage infections of the experimental malaria Plasmodium chabaudi. Blood-stage malaria induces extramedullary erythropoiesis in the liver. This study investigates how vaccination affects the course of malaria-induced expression of erythrocytic genes in the liver. METHODS: Female Balb/c mice were vaccinated at week 3 and week 1 before challenging with 106 P. chabaudi-parasitized erythrocytes. The non-infectious vaccine consisted of erythrocyte ghosts isolated from P. chabaudi-infected erythrocytes. Gene expression microarrays and quantitative real-time PCR were used to compare mRNA expression of different erythrocytic genes in the liver of vaccination-protected and non-protected mice during infections on days 0, 1, 4, 8, and 11 p.i. RESULTS: Global transcriptomics analyses reveal vaccination-induced modifications of malaria-induced increases in hepatic gene expression on days 4 and 11 p.i. On these days, vaccination also alters hepatic expression of the erythropoiesis-involved genes Ermap, Kel, Rhd, Rhag, Slc4a1, Gypa, Add2, Ank1, Epb4.1, Epb4.2, Epb4.9, Spta1, Sptb, Tmod1, Ahsp, Acyp1, Gata1, Gfi1b, Tal1, Klf1, Epor, and Cldn13. In vaccination-protected mice, expression of these genes, except Epb4.1, is significantly higher on day 4 p.i. than in un-protected non-vaccinated mice, reaches maximal expression at peak parasitaemia on day 8 p.i., and is slowed down or even decreased towards the end of crisis phase on day 11 p.i.. After day 1 p.i., Epor expression takes about the same course as that of the other erythroid genes. Hepatic expression of Epo, however, is delayed in both vaccinated and non-vaccinated mice for the first 4 days p.i. and is maximal at significantly higher levels in vaccinated mice on day 8 p.i., before declining towards the end of crisis phase on day 11 p.i. CONCLUSION: The present data indicate that vaccination accelerates malaria-induced erythroblastosis in the liver for 1-2 days. This may contribute to earlier replenishment of peripheral red blood cells by liver-derived reticulocytes, which may favour final survival of otherwise lethal blood-stage malaria, since reticulocytes are not preferred as host cells by P. chabaudi.

Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite.

BACKGROUND: Human blood cells (erythrocytes) serve as hosts for the malaria parasite Plasmodium falciparum during its 48-h intraerythrocytic developmental cycle (IDC). Established in vitro protocols allow for the study of host-parasite interactions during this phase and, in particular, high-resolution metabolomics can provide a window into host-parasite interactions that support parasite development. METHODS: Uninfected and parasite-infected erythrocyte cultures were maintained at 2% haematocrit for the duration of the IDC, while parasitaemia was maintained at 7% in the infected cultures. The parasite-infected cultures were synchronized to obtain stage-dependent information of parasite development during the IDC. Samples were collected in quadruplicate at six time points from the uninfected and parasite-infected cultures and global metabolomics was used to analyse cell fractions of these cultures. RESULTS: In uninfected and parasite-infected cultures during the IDC, 501 intracellular metabolites, including 223 lipid metabolites, were successfully quantified. Of these, 19 distinct metabolites were present only in the parasite-infected culture, 10 of which increased to twofold in abundance during the IDC. This work quantified approximately five times the metabolites measured in previous studies of similar research scope, which allowed for more detailed analyses. Enrichment in lipid metabolism pathways exhibited a time-dependent association with different classes of lipids during the IDC. Specifically, enrichment occurred in sphingolipids at the earlier stages, and subsequently in lysophospholipid and phospholipid metabolites at the intermediate and end stages of the IDC, respectively. In addition, there was an accumulation of 18-, 20-, and 22-carbon polyunsaturated fatty acids, which produce eicosanoids and promote gametocytogenesis in infected erythrocyte cultures. CONCLUSIONS: The current study revealed a number of heretofore unidentified metabolic components of the host-parasite system, which the parasite may exploit in a time-dependent manner to grow over the course of its development in the blood stage. Notably, the analyses identified components, such as precursors of immunomodulatory molecules, stage-dependent lipid dynamics, and metabolites, unique to parasite-infected cultures. These conclusions are reinforced by the metabolic alterations that were characterized during the IDC, which were in close agreement with those known from previous studies of blood-stage infection.

DSM265 at 400 Milligrams Clears Asexual Stage Parasites but Not Mature Gametocytes from the Blood of Healthy Subjects Experimentally Infected with Plasmodium falciparum.

DSM265 is a novel antimalarial drug in clinical development that acts as a selective inhibitor of Plasmodium dihydroorotate dehydrogenase. In a previous phase 1b study, a single 150-mg dose of DSM265 showed partial efficacy against experimentally induced blood-stage Plasmodium falciparum malaria (IBSM). Pharmacokinetic/pharmacodynamic modeling predicted a human efficacious dose of 340 mg. The primary objectives of the current study were to determine the safety and efficacy of a single oral 400-mg dose of DSM265 against P. falciparum in the IBSM model. Eight healthy participants were inoculated intravenously with 2,800 parasites and treated with DSM265 7 days later. Unexpectedly, one participant did not develop parasitemia during the study. All other participants developed parasitemia, with the complete clearance of asexual parasites occurring following DSM265 treatment. All seven subjects also became gametocytemic. The secondary objectives were to investigate the gametocytocidal and transmission-blocking activity of a second 400-mg dose of DSM265, which was administered 23 days after inoculation. Gametocytes were not cleared by the second dose of DSM265, and transmission-blocking activity could not be determined due to low gametocyte densities. Three DSM265-related adverse events occurred, including a cutaneous rash in one subject on the day of the second DSM265 dose. The results obtained in this study support the prediction of the efficacious dose of DSM265 and provide further evidence that DSM265 is generally safe and well tolerated. In addition, this study confirms preclinical data indicating that DSM265 permits the development and maturation of gametocytes and does not clear mature circulating gametocytes. (This study has been registered at ClinicalTrials.gov under identifier NCT02573857.).

RTS,S/AS01E immunization increases antibody responses to vaccine-unrelated Plasmodium falciparum antigens associated with protection against clinical malaria in African children: a case-control study.

BACKGROUND: Vaccination and naturally acquired immunity against microbial pathogens may have complex interactions that influence disease outcomes. To date, only vaccine-specific immune responses have routinely been investigated in malaria vaccine trials conducted in endemic areas. We hypothesized that RTS,S/A01E immunization affects acquisition of antibodies to Plasmodium falciparum antigens not included in the vaccine and that such responses have an impact on overall malaria protective immunity. METHODS: We evaluated IgM and IgG responses to 38 P. falciparum proteins putatively involved in naturally acquired immunity to malaria in 195 young children participating in a case-control study nested within the African phase 3 clinical trial of RTS,S/AS01E (MAL055 NCT00866619) in two sites of different transmission intensity (Kintampo high and Manhiça moderate/low). We measured antibody levels by quantitative suspension array technology and applied regression models, multimarker analysis, and machine learning techniques to analyze factors affecting their levels and correlates of protection. RESULTS: RTS,S/AS01E immunization decreased antibody responses to parasite antigens considered as markers of exposure (MSP142, AMA1) and levels correlated with risk of clinical malaria over 1-year follow-up. In addition, we show for the first time that RTS,S vaccination increased IgG levels to a specific group of pre-erythrocytic and blood-stage antigens (MSP5, MSP1 block 2, RH4.2, EBA140, and SSP2/TRAP) which levels correlated with protection against clinical malaria (odds ratio [95% confidence interval] 0.53 [0.3-0.93], p = 0.03, for MSP1; 0.52 [0.26-0.98], p = 0.05, for SSP2) in multivariable logistic regression analyses. CONCLUSIONS: Increased antibody responses to specific P. falciparum antigens in subjects immunized with this partially efficacious vaccine upon natural infection may contribute to overall protective immunity against malaria. Inclusion of such antigens in multivalent constructs could result in more efficacious second-generation multistage vaccines.

From marginal to essential: the golden thread between nutrient sensing, medium composition and Plasmodium vivax maturation in in vitro culture.

Historically neglected, due to its biological peculiarities, the absence of a continuous long-term in vitro blood stage culture system and a propensity towards high morbidity rather than mortality, Plasmodium vivax was put back on the agenda during the last decade by the paradigm shift in the fight against malaria from malaria control to malaria eradication. While the incidence of the deadliest form of malaria, Plasmodium falciparum malaria, has declined since this paradigm shift took hold, the prospects of eradication are now threatened by the increase in the incidence of other human malaria parasite species. Plasmodium vivax is geographically the most widely distributed human malaria parasite, characterized by millions of clinical cases every year and responsible for a massive economic burden. The urgent need to tackle the unique biological challenges posed by this parasite led to renewed efforts aimed at establishing a continuous, long-term in vitro P. vivax blood stage culture. Based on recent discoveries on the role of nutrient sensing in Plasmodium's pathophysiology, this review article critically assesses the extensive body of literature concerning Plasmodium culture conditions with a specific focus on culture media used in attempts to culture different Plasmodium spp. Hereby, the effect of specific media components on the parasite's in vitro fitness and the maturation of the parasite's host cell, the reticulocyte, is analysed. Challenging the wide-held belief that it is sufficient to find the right parasite isolate and give it the right type of cells to invade for P. vivax to grow in vitro, this review contends that a healthy side-by-side maturation of both the parasite and its host cell, the reticulocyte, is necessary in the adaptation of P. vivax to in vitro growth and argues that culture conditions and the media in particular play an essential role in this maturation process.

A step forward for an attenuated blood-stage malaria vaccine.

Efforts to develop an effective malaria vaccine have encountered multiple challenges, and have had limited success to date. As the need remains urgent, novel approaches must be explored. One concept that has gained attention uses whole malaria parasites. Building on preclinical studies in animal models, Stanisic et al. describe the development of a vaccine based on chemically attenuated Plasmodium falciparum blood-stage parasites, with an evaluation of safety and immunogenicity in malaria-naïve human subjects. The vaccine was shown to be safe, well tolerated, and capable of priming antigen-specific T cells. This work, and the completion of an initial clinical trial in human subjects, represents a significant advance. While the path forward for this attenuated vaccine remains challenging, these initial findings are encouraging. Importantly, the results provide the foundation and framework for testing modified immunization protocols, and designing subsequent clinical trials to further evaluate safety, test for enhanced immunogenicity, and ultimately measure protective efficacy.Please see related article: https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-018-1173-9.

Vitamin D receptor regulates intestinal inflammatory response in mice infected with blood stage malaria.

Malaria is a harmful disease affecting both tropical and subtropical countries and causing sometimes fatal complications. The effects of malaria-related complications on the intestine have been relatively neglected, and the reasons for the intestinal damage caused by malaria infection are not yet clear. The present study aims to evaluate the influence of intestinal vitamin D receptor on host-pathogen interactions during malaria induced in mice by Plasmodium chabaudi. To induce the infection, animals were infected with 106P. chabaudi-parasitized erythrocytes. Mice were sacrificed on day 8 post-infection. The infected mice experienced a significant body weight loss and parasitaemia affecting about 46% of RBCs. Infection caused marked pathological changes in the intestinal tissue indicated by shortening of the intestine and villi. Moreover, the phagocytic activity of macrophages increased significantly (P < 0.01) in the infected villi compared to the non-infected ones. Infection by the parasite also induced marked upregulation of nuclear factor-kappa B, inducible nitric oxide synthase, Vitamin D Receptor, interleukin-1ß, tumour necrosis factor alpha and interferon gamma-mRNA. It can be implied from this that vitamin D receptor has a role in regulating malarial infection.