Publications by authors named "Robert W Sauerwein"

207 Publications

Infection Manager System (IMS) as a new hemocytometry-based bacteremia detection tool: A diagnostic accuracy study in a malaria-endemic area of Burkina Faso.

PLoS Negl Trop Dis 2021 Mar 1;15(3):e0009187. Epub 2021 Mar 1.

Nijmegen Institute of International Health, Radboudumc, Nijmegen, the Netherlands.

Background: New hemocytometric parameters can be used to differentiate causes of acute febrile illness (AFI). We evaluated a software algorithm-Infection Manager System (IMS)-which uses hemocytometric data generated by Sysmex hematology analyzers, for its accuracy to detect bacteremia in AFI patients with and without malaria in Burkina Faso. Secondary aims included comparing the accuracy of IMS with C-reactive protein (CRP) and procalcitonin (PCT).

Methods: In a prospective observational study, patients of ≥ three-month-old (range 3 months- 90 years) presenting with AFI were enrolled. IMS, blood culture and malaria diagnostics were done upon inclusion and additional diagnostics on clinical indication. CRP, PCT, viral multiplex PCR on nasopharyngeal swabs and bacterial- and malaria PCR were batch-tested retrospectively. Diagnostic classification was done retrospectively using all available data except IMS, CRP and PCT results.

Findings: A diagnosis was affirmed in 549/914 (60.1%) patients and included malaria (n = 191) bacteremia (n = 69), viral infections (n = 145), and malaria-bacteremia co-infections (n = 47). The overall sensitivity, specificity, and negative predictive value (NPV) of IMS for detection of bacteremia in patients of ≥ 5 years were 97.0% (95% CI: 89.8-99.6), 68.2% (95% CI: 55.6-79.1) and 95.7% (95% CI: 85.5-99.5) respectively, compared to 93.9% (95% CI: 85.2-98.3), 39.4% (95% CI: 27.6-52.2), and 86.7% (95% CI: 69.3-96.2) for CRP at ≥20mg/L. The sensitivity, specificity and NPV of PCT at 0.5 ng/ml were lower at respectively 72.7% (95% CI: 60.4-83.0), 50.0% (95% CI: 37.4-62.6) and 64.7% (95% CI: 50.1-77.6) The diagnostic accuracy of IMS was lower among malaria cases and patients <5 years but remained equal to- or higher than the accuracy of CRP.

Interpretation: IMS is a new diagnostic tool to differentiate causes of AFI. Its high NPV for bacteremia has the potential to improve antibiotic dispensing practices in healthcare facilities with hematology analyzers. Future studies are needed to evaluate whether IMS, combined with malaria diagnostics, may be used to rationalize antimicrobial prescription in malaria endemic areas.

Trial Registration: ClinicalTrials.gov (NCT02669823) https://clinicaltrials.gov/ct2/show/NCT02669823.
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http://dx.doi.org/10.1371/journal.pntd.0009187DOI Listing
March 2021

Systematic Identification of Plasmodium Falciparum Sporozoite Membrane Protein Interactions Reveals an Essential Role for the p24 Complex in Host Infection.

Mol Cell Proteomics 2021 Jan 27;20:100038. Epub 2021 Jan 27.

Cell Surface Signalling Laboratory, Wellcome Sanger Institute, Cambridge, United Kingdom; Malaria Programme, Wellcome Sanger Institute, Cambridge, United Kingdom; Department of Biology, Hull York Medical School, York Biomedical Research Institute, University of York, York, United Kingdom. Electronic address:

Sporozoites are a motile form of malaria-causing Plasmodium falciparum parasites that migrate from the site of transmission in the dermis through the bloodstream to invade hepatocytes. Sporozoites interact with many cells within the host, but the molecular identity of these interactions and their role in the pathology of malaria is poorly understood. Parasite proteins that are secreted and embedded within membranes are known to be important for these interactions, but our understanding of how they interact with each other to form functional complexes is largely unknown. Here, we compile a library of recombinant proteins representing the repertoire of cell surface and secreted proteins from the P. falciparum sporozoite and use an assay designed to detect extracellular interactions to systematically identify complexes. We identify three protein complexes including an interaction between two components of the p24 complex that is involved in the trafficking of glycosylphosphatidylinositol-anchored proteins through the secretory pathway. Plasmodium parasites lacking either gene are strongly inhibited in the establishment of liver-stage infections. These findings reveal an important role for the p24 complex in malaria pathogenesis and show that the library of recombinant proteins represents a valuable resource to investigate P. falciparum sporozoite biology.
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http://dx.doi.org/10.1074/mcp.RA120.002432DOI Listing
January 2021

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

EMBO J 2021 Jan 18:e106583. Epub 2021 Jan 18.

Radboudumc Center for Infectious Diseases, Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.

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.
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http://dx.doi.org/10.15252/embj.2020106583DOI Listing
January 2021

Controlled Human Malaria Infection Induces Long-Term Functional Changes in Monocytes.

Front Mol Biosci 2020 26;7:604553. Epub 2020 Nov 26.

Department of Medical Microbiology and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

Innate immune memory responses (also termed "") have been described in monocytes after BCG vaccination and after stimulation with microbial and endogenous ligands such as LPS, β-glucan, oxidized LDL, and monosodium urate crystals. However, whether clinical infections are also capable of inducing a trained immunity phenotype remained uncertain. We evaluated whether infection can induce innate immune memory by measuring monocyte-derived cytokine production from five volunteers undergoing Controlled Human Malaria Infection. Monocyte responses followed a biphasic pattern: during acute infection, monocytes produced lower amounts of inflammatory cytokines upon secondary stimulation, but 36 days after malaria infection they produced significantly more IL-6 and TNF-α in response to various stimuli. Furthermore, transcriptomic and epigenomic data analysis revealed a clear reprogramming of monocytes at both timepoints, with long-term changes of H3K4me3 at the promoter regions of inflammatory genes that remain present for several weeks after parasite clearance. These findings demonstrate an epigenetic basis of trained immunity induced by human malaria .
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http://dx.doi.org/10.3389/fmolb.2020.604553DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726436PMC
November 2020

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Immunity 2020 Oct 17;53(4):733-744.e8. Epub 2020 Sep 17.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.
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http://dx.doi.org/10.1016/j.immuni.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572793PMC
October 2020

Generation of Novel NF135 and NF54 Lines Expressing Fluorescent Reporter Proteins Under the Control of Strong and Constitutive Promoters.

Front Cell Infect Microbiol 2020 10;10:270. Epub 2020 Jun 10.

Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different ( ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard NF54 background and in a recently described Cambodian NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to NF54. We first generated NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (, PF3D7_1243600) and 40S ribosomal protein S30 (, PF3D7_0219200). We then generated and characterized reporter lines in the NF135.C10 line which express GFP driven by the and promoters as well as by the previously used α promoter (α, ). The reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the promoter valuable novel tools for analyses of .
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http://dx.doi.org/10.3389/fcimb.2020.00270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298075PMC
June 2020

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

Sci Transl Med 2020 05;12(544)

Radboudumc Center for Infectious Diseases, Department of Medical Microbiology, Radboud University Medical Center, 6525 GA Nijmegen, Netherlands.

Immunization with attenuated sporozoites can induce protection against malaria infection, as shown by (Pf) sporozoites attenuated by radiation in multiple clinical trials. As alternative attenuation strategy with a more homogeneous population of Pf sporozoites (PfSPZ), genetically engineered 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∆∆), 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 × 10 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 × 10 or 4.5 × 10 PfSPZ, = 13 each), PfSPZ Vaccine (radiation-attenuated PfSPZ, 4.5 × 10 PfSPZ, = 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.
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http://dx.doi.org/10.1126/scitranslmed.aaz5629DOI Listing
May 2020

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

Sci Transl Med 2020 05;12(544)

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.

For some diseases, successful vaccines have been developed using a nonpathogenic counterpart of the causative microorganism of choice. The nonpathogenicity of the rodent () parasite in humans prompted us to evaluate its potential as a platform for vaccination against human infection by (), a causative agent of malaria. We hypothesized that the genetic insertion of a leading protein target for clinical development of a malaria vaccine, circumsporozoite protein (CSP), in its natural pre-erythrocytic environment, would enhance 's capacity to induce protective immunity against infection. Hence, we recently generated a transgenic sporozoite immunization platform expressing CSP (Vac), and we now report the clinical evaluation of its biological activity against controlled human malaria infection (CHMI). This first-in-human trial shows that Vac is safe and well tolerated, when administered by a total of ~300 Vac-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, = 0.03) and decreased parasite density were observed after immunization, corresponding to an estimated 95% reduction in liver parasite burden (confidence interval, 56 to 99%; = 0.010). Vac elicits dose-dependent cross-species cellular immune responses and functional CSP-dependent antibody responses that efficiently block sporozoite invasion of liver cells in vitro. This study demonstrates that Vac immunization elicits a marked biological effect, inhibiting a subsequent infection by the human parasite, and establishes the clinical validation of a new paradigm in malaria vaccination.
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http://dx.doi.org/10.1126/scitranslmed.aay2578DOI Listing
May 2020

Activatory Receptor NKp30 Predicts NK Cell Activation During Controlled Human Malaria Infection.

Front Immunol 2019 10;10:2864. Epub 2019 Dec 10.

Department of Medical Microbiology and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

Natural killer (NK) cells are known to be activated during malaria infection, exhibiting both cytokine production and cytotoxic functions. However, NK cells are heterogeneous in their expression of surface activatory and inhibitory receptors which may influence their response to malaria parasites. Here, we studied the surface marker profile and activation dynamics of NK cells during a Controlled Human Malaria Infection in 12 healthy volunteers. Although there was significant inter-patient variability in timing and magnitude of NK cell activation, we found a consistent and strong increase in expression of the activatory receptor NKp30. Moreover, high baseline NKp30 expression was associated with NK cell activation at lower parasite densities. Our data suggest that NKp30 expression may influence the NK cell response to , explaining inter-patient heterogeneity and suggesting a functional role for this receptor in malaria.
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http://dx.doi.org/10.3389/fimmu.2019.02864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916516PMC
November 2020

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential.

Genome Med 2020 01 8;12(1). Epub 2020 Jan 8.

Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.

Background: Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.

Methods: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia.

Results: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8 T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region.

Conclusions: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.
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http://dx.doi.org/10.1186/s13073-019-0708-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950926PMC
January 2020

Fast and fierce versus slow and smooth: Heterogeneity in immune responses to Plasmodium in the controlled human malaria infection model.

Immunol Rev 2020 01 12;293(1):253-269. Epub 2019 Oct 12.

Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.

Controlled human malaria infection (CHMI) is an established model in clinical malaria research. Upon exposure to Plasmodium falciparum parasites, malaria-naive volunteers differ in dynamics and composition of their immune profiles and subsequent capacity to generate protective immunity. CHMI volunteers are either inflammatory responders who have prominent cellular IFN-γ production primarily driven by adaptive T cells, or tempered responders who skew toward antibody-mediated humoral immunity. When exposed to consecutive CHMIs under antimalarial chemoprophylaxis, individuals who can control parasitemia after a single immunization (fast responders) are more likely to be protected against a subsequent challenge infection. Fast responders tend to be inflammatory responders who can rapidly induce long-lived IFN-γ T cell responses. Slow responders or even non-responders can also be protected, but via a more diverse range of responses that take a longer time to reach full protective efficacy, in part due to their tempered phenotype. The latter group can be identified at baseline before CHMI by higher expression of inhibitory ligands CTLA-4 and TIM-3 on CD4 T cells. Delineating heterogeneity in human immune responses to P. falciparum will facilitate rational design and strategy towards effective malaria vaccines.
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http://dx.doi.org/10.1111/imr.12811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6973142PMC
January 2020

Antimalarial pantothenamide metabolites target acetyl-coenzyme A biosynthesis in .

Sci Transl Med 2019 09;11(510)

TropIQ Health Sciences, Nijmegen, Netherlands.

Malaria eradication is critically dependent on new therapeutics that target resistant parasites and block transmission of the disease. Here, we report that pantothenamide bioisosteres were active against blood-stage parasites and also blocked transmission of sexual stages to the mosquito vector. These compounds were resistant to degradation by serum pantetheinases, showed favorable pharmacokinetic properties, and cleared parasites in a humanized mouse model of infection. Metabolomics revealed that coenzyme A biosynthetic enzymes converted pantothenamides into coenzyme A analogs that interfered with parasite acetyl-coenzyme A anabolism. Resistant parasites generated in vitro showed mutations in acetyl-coenzyme A synthetase and acyl-coenzyme A synthetase 11. Introduction and reversion of these mutations in using CRISPR-Cas9 gene editing confirmed the roles of these enzymes in the sensitivity of the malaria parasites to pantothenamides. These pantothenamide compounds with a new mode of action may have potential as drugs against malaria parasites.
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http://dx.doi.org/10.1126/scitranslmed.aas9917DOI Listing
September 2019

A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle.

mBio 2019 09 17;10(5). Epub 2019 Sep 17.

Signalling in Apicomplexan Parasites Laboratory, The Francis Crick Institute, London, United Kingdom

has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1. One of the major limitations in studying is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.
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http://dx.doi.org/10.1128/mBio.01170-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751054PMC
September 2019

Transforming growth factor-beta profiles correlate with clinical symptoms and parameters of haemostasis and inflammation in a controlled human malaria infection.

Cytokine 2020 01 13;125:154838. Epub 2019 Sep 13.

Department of Medical Microbiology & Infectious Diseases, Erasmus MC University Medical Center, Rotterdam 3015 GD, the Netherlands. Electronic address:

Background: After a controlled human malaria infection (CHMI), presentation of clinical signs and symptoms and host responses is heterogeneous. Transforming growth factor-beta (TGF-β) is the first serum cytokine that changes in malaria-naïve volunteers after CHMI. We studied a possible relation between TGF-β changes, pro-inflammatory cytokines, activation of haemostasis and endothelial cells and clinical symptoms.

Methods: A panel of cytokines including TGF-β, and markers of activation of haemostasis and endothelial cells were measured in blood samples of 15 volunteers at baseline before CHMI and during CHMI at day of treatment. The change of the parameters on the day of treatment was examined for a significant alteration during infection.

Results: Nine of 15 volunteers showed a significant decrease in TGF-β compared to baseline, with concomitant increased concentrations of D-dimer (p = 0.012), Von Willebrand factor (p = 0.017), IL-6 (p = 0.012) and IFN-γ (0.028) and a significantly decreased platelet count (p = 0.011). In contrast, 6 of 15 volunteers showed sustained or increased TGF-β concentrations without change in the aforementioned parameters. The sustained responders presented with less moderate and severe clinical symptoms than the negative responders (p = 0.036) and had a higher baseline lymphocyte count (p = 0.026). TGF-β concentrations did not correlate with the parasitaemia on day of treatment.

Conclusion: Early decreases of serum TGF-β might function a marker for a pro-inflammatory host response and downstream clinical symptoms and pathology during CHMI.
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http://dx.doi.org/10.1016/j.cyto.2019.154838DOI Listing
January 2020

s230 and s48/45 Fusion Proteins Elicit Strong Transmission-Blocking Antibody Responses Against .

Front Immunol 2019 5;10:1256. Epub 2019 Jun 5.

Department for Congenital Disorders, Statens Serum Institut, Copenhagen, Denmark.

The s230 and s48/45 proteins are expressed during transmission from man to mosquito and are leading candidates for a malaria transmission blocking vaccine. Individually they generate transmission blocking (TB) antibodies in rodent models. Whether the single protein vaccines are suitable to use in field settings will primarily depend on their potency to elicit functional antibodies. We hypothesized that a combination of both proteins will be more potent than each protein individually. Therefore we designed chimeric proteins composed of fragments of both s230 and s48/45 as well as single protein fragments, and expressed these in . Both the individual s230 and s48/45 fragments and chimeras elicited high levels of functional antibodies in mice. Importantly, one of the chimeric proteins elicited over threefold higher transmission blocking antibody responses than the single antigens alone. Furthermore the immunogenicity of one of the chimeras could be enhanced through coupling to a virus-like particle (VLP). Altogether these data support further clinical development of these novel constructs.
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http://dx.doi.org/10.3389/fimmu.2019.01256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6560166PMC
October 2020

Whole-blood transcriptomic signatures induced during immunization by chloroquine prophylaxis and Plasmodium falciparum sporozoites.

Sci Rep 2019 06 10;9(1):8386. Epub 2019 Jun 10.

Department of Medical Microbiology and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands.

A highly effective vaccine that confers sterile protection to malaria is urgently needed. Immunization under chemoprophylaxis with sporozoites (CPS) consistently confers high levels of protection in the Controlled Human Malaria infection (CHMI) model. To provide a broad, unbiased assessment of the composition and kinetics of direct ex vivo human immune responses to CPS, we profiled whole-blood transcriptomes by RNA-seq before and during CPS immunization and following CHMI challenge. Differential expression of genes enriched in modules related to T cells, NK cells, protein synthesis, and mitochondrial processes were detected in fully protected individuals four weeks after the first immunization. Non-protected individuals demonstrated transcriptomic changes after the third immunization and the day of treatment, with upregulation of interferon and innate inflammatory genes and downregulation of B-cell signatures. Protected individuals demonstrated more significant interactions between blood transcription modules compared to non-protected individuals several weeks after the second and third immunizations. These data provide insight into the molecular and cellular basis of CPS-induced immune protection from P. falciparum infection.
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http://dx.doi.org/10.1038/s41598-019-44924-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557840PMC
June 2019

T1-Polarized T Cells Delay Naturally-Acquired Immunity to Malaria.

Front Immunol 2019 17;10:1096. Epub 2019 May 17.

Department of Medical Microbiology, RadboudUMC Centre for Infectious Diseases, Nijmegen, Netherlands.

Humoral immunity is a critical effector arm for protection against malaria but develops only slowly after repeated infections. T cell-mediated regulatory dynamics affect the development of antibody responses to parasites. Here, we hypothesize that T follicular helper cell (T) polarization generated by repeated asexual blood-stage infections delays the onset of protective humoral responses. IFN-γ production promotes polarization toward T1 and increased generation of regulatory follicular helper cells (T). Delineating the mechanisms that drive T1 polarization will provide clues for appropriate induction of lasting, protective immunity against malaria.
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http://dx.doi.org/10.3389/fimmu.2019.01096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533880PMC
June 2020

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

BMC Med 2019 05 31;17(1):103. Epub 2019 May 31.

Nijmegen Institute of International Health, Radboud University Medical Centre, Nijmegen, the Netherlands.

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 (R = 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).
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http://dx.doi.org/10.1186/s12916-019-1334-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543632PMC
May 2019

A NF54 Reporter Line Expressing mCherry-Luciferase in Gametocytes, Sporozoites, and Liver-Stages.

Front Cell Infect Microbiol 2019 16;9:96. Epub 2019 Apr 16.

Department of Parasitology, Leiden University Medical Center, Leiden, Netherlands.

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic (Pf) NF54 line was generated that expresses a fusion of and genes under the control of the .3 gene promoter (line mCherry-luc@etramp10.3). .3 is related to rodent and the promoter has been used to drive high transgene expression in rodent parasite sporozoites and liver-stages. We examined transgene expression throughout the complete life cycle and compared this expression to transgenic lines expressing mCherry-luciferase and GFP-luciferase under control of the constitutive and promoters. The parasites express mCherry in gametocytes, sporozoites, and liver-stages. While no mCherry signal was detected in asexual blood-stage parasites above background levels, luciferase expression was detected in asexual blood-stages, as well as in gametocytes, sporozoites and liver-stages, with the highest levels of reporter expression detected in stage III-V gametocytes and in sporozoites. The expression of mCherry and luciferase in gametocytes and sporozoites makes this transgenic parasite line suitable to use in assays that examine the effect of transmission blocking inhibitors and to analyse gametocyte and sporozoite biology.
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http://dx.doi.org/10.3389/fcimb.2019.00096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477837PMC
December 2019

Author Correction: The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission.

Sci Rep 2019 May 3;9(1):7061. Epub 2019 May 3.

Leiden Malaria Research Group, Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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http://dx.doi.org/10.1038/s41598-019-43505-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499769PMC
May 2019

Outcomes of controlled human malaria infection after BCG vaccination.

Nat Commun 2019 02 20;10(1):874. Epub 2019 Feb 20.

Department of Medical Microbiology, Radboud University Medical Center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands.

Recent evidence suggests that certain vaccines, including Bacillus-Calmette Guérin (BCG), can induce changes in the innate immune system with non-specific memory characteristics, termed 'trained immunity'. Here we present the results of a randomised, controlled phase 1 clinical trial in 20 healthy male and female volunteers to evaluate the induction of immunity and protective efficacy of the anti-tuberculosis BCG vaccine against a controlled human malaria infection. After malaria challenge infection, BCG vaccinated volunteers present with earlier and more severe clinical adverse events, and have significantly earlier expression of NK cell activation markers and a trend towards earlier phenotypic monocyte activation. Furthermore, parasitemia in BCG vaccinated volunteers is inversely correlated with increased phenotypic NK cell and monocyte activation. The combined data demonstrate that BCG vaccination alters the clinical and immunological response to malaria, and form an impetus to further explore its potential in strategies for clinical malaria vaccine development.
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http://dx.doi.org/10.1038/s41467-019-08659-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382772PMC
February 2019

Antibody Biomarkers Associated with Sterile Protection Induced by Controlled Human Malaria Infection under Chloroquine Prophylaxis.

mSphere 2019 02 20;4(1). Epub 2019 Feb 20.

Vaccine Research and Development Center, Department of Physiology and Biophysics, University of California, Irvine, Irvine, California, USA.

Immunization with sporozoites under chloroquine chemoprophylaxis (CPS) induces distinctly preerythrocytic and long-lasting sterile protection against homologous controlled human malaria infection (CHMI). To identify possible humoral immune correlates of protection, plasma samples were collected from 38 CPS-immunized Dutch volunteers for analysis using a whole proteome microarray with 7,455 full-length or segmented protein features displaying about 91% of the total proteome. We identified 548 reactive antigens representing 483 unique proteins. Using the breadth of antibody responses for each subject in a mixture-model algorithm, we observed a trimodal pattern, with distinct groups of 16 low responders, 19 medium responders, and 3 high responders. Fifteen out of 16 low responders, 12 of the 19 medium responders, and 3 out of 3 high responders were fully protected from a challenge infection. In the medium-responder group, we identified six novel antigens associated with protection (area under the curve [AUC] value of ≥0.75;  < 0.05) and six other antigens that were specifically increased in nonprotected volunteers (AUC value of ≤0.25;  < 0.05). When used in combination, the multiantigen classifier predicts CPS-induced protective efficacy with 83% sensitivity and 88% specificity. The antibody response patterns characterized in this study represent surrogate markers that may provide rational guidance for clinical vaccine development. Infection by parasites has been a major cause of mortality and morbidity in humans for thousands of years. Despite the considerable reduction of deaths, according to the WHO, over 5 billion people are still at risk, with about 216 million worldwide cases occurring in 2016. More compelling, 15 countries in sub-Saharan Africa bore 80% of the worldwide malaria burden. Complete eradication has been challenging, and the development of an affordable and effective vaccine will go a long way in achieving elimination. However, identifying vaccine candidate targets has been difficult. In the present study, we use a highly effective immunization protocol that confers long-lasting sterile immunity in combination with a whole proteome microarray to identify antibody responses associated with protection. This study characterizes a novel antibody profile associated with sterile protective immunity and trimodal humoral responses that sheds light on the possible mechanism of CPS-induced immunity against parasites.
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http://dx.doi.org/10.1128/mSphereDirect.00027-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6382972PMC
February 2019

Can Patrolling Liver-Resident T Cells Control Human Malaria Parasite Development?

Trends Immunol 2019 03 31;40(3):186-196. Epub 2019 Jan 31.

Department of Medical Microbiology, Radboud University Medical Center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands; Radboud Center for Infectious Diseases, Radboud University Medical Center, PO Box 9101, 6500 HB, Nijmegen, The Netherlands. Electronic address:

Recently, a population of non-recirculating, tissue-resident memory CD8 T cells has been identified; cells that seems to act as key sentinels for invading microorganisms with enhanced effector functions. In malaria, the liver represents the first site for parasite development before a definite infection is established in circulating red blood cells. Here, we discuss the evidence obtained from animal models on several diseases and hypothesize that liver-resident memory CD8 T cells (hepatic T) play a critical role in providing protective liver-stage immunity against Plasmodium malaria parasites. Although observations in human malaria trials are limited to peripheral blood, we propose recommendations for the translation of some of these findings to human malaria research.
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http://dx.doi.org/10.1016/j.it.2019.01.002DOI Listing
March 2019

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

NPJ Vaccines 2018 27;3:54. Epub 2018 Nov 27.

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.

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 () that expresses the () circumsporozoite protein (CS), and showed that this parasite line (Vac) was capable of (1) infecting and developing in human hepatocytes but not in human erythrocytes, and (2) inducing neutralizing antibodies against the human parasite. Here, we analyzed Vac in detail and developed tools necessary for its use in clinical studies. A microbiological contaminant-free Master Cell Bank of Vac 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 Vac 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 Vac-related toxicity. In vivo drug sensitivity assays employing rodent models of infection showed that both the liver and the blood stage forms of Vac were completely eliminated by Malarone treatment. Collectively, our pre-clinical safety assessment demonstrates that Vac possesses all characteristics necessary to advance into clinical evaluation.
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http://dx.doi.org/10.1038/s41541-018-0091-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258718PMC
November 2018

Structural delineation of potent transmission-blocking epitope I on malaria antigen Pfs48/45.

Nat Commun 2018 10 26;9(1):4458. Epub 2018 Oct 26.

Program in Molecular Medicine, The Hospital for Sick Children Research Institute, Toronto, M5G 0A4, ON, Canada.

Interventions that can block the transmission of malaria-causing Plasmodium falciparum (Pf) between the human host and Anopheles vector have the potential to reduce the incidence of malaria. Pfs48/45 is a gametocyte surface protein critical for parasite development and transmission, and its targeting by monoclonal antibody (mAb) 85RF45.1 leads to the potent reduction of parasite transmission. Here, we reveal how the Pfs48/45 6C domain adopts a (SAG1)-related-sequence (SRS) fold. We structurally delineate potent epitope I and show how mAb 85RF45.1 recognizes an electronegative surface with nanomolar affinity. Analysis of Pfs48/45 sequences reveals that polymorphisms are rare for residues involved at the binding interface. Humanization of rat-derived mAb 85RF45.1 conserved the mode of recognition and activity of the parental antibody, while also improving its thermostability. Our work has implications for the development of transmission-blocking interventions, both through improving vaccine designs and the testing of passive delivery of mAbs in humans.
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http://dx.doi.org/10.1038/s41467-018-06742-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203815PMC
October 2018

The Plasmodium falciparum male gametocyte protein P230p, a paralog of P230, is vital for ookinete formation and mosquito transmission.

Sci Rep 2018 10 8;8(1):14902. Epub 2018 Oct 8.

Leiden Malaria Research Group, Parasitology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes. In rodent malaria parasites this protein is dispensable throughout the complete life-cycle; however, its function in P. falciparum is unknown. Using CRISPR/Cas9 methodology we disrupted the gene encoding Pfp230p resulting in P. falciparum mutants (PfΔp230p) lacking P230p expression. The PfΔp230p mutants produced normal numbers of male and female gametocytes, which retained expression of P48/45 and P230. Upon activation male PfΔp230p gametocytes undergo exflagellation and form male gametes. However, male gametes are unable to attach to red blood cells resulting in the absence of characteristic exflagellation centres in vitro. In the absence of P230p, zygote formation as well as oocyst and sporozoite development were strongly reduced (>98%) in mosquitoes. These observations demonstrate that P230p, like P230 and P48/45, has a vital role in P. falciparum male fertility and zygote formation and warrants further investigation as a potential transmission blocking vaccine candidate.
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http://dx.doi.org/10.1038/s41598-018-33236-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175877PMC
October 2018

Liver Injury in Uncomplicated Malaria is an Overlooked Phenomenon: An Observational Study.

EBioMedicine 2018 Oct 19;36:131-139. Epub 2018 Sep 19.

Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, the Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.ebiom.2018.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197763PMC
October 2018

Structural basis for recognition of the malaria vaccine candidate Pfs48/45 by a transmission blocking antibody.

Nat Commun 2018 09 20;9(1):3822. Epub 2018 Sep 20.

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

The quest to develop an effective malaria vaccine remains a major priority in the fight against global infectious disease. An approach with great potential is a transmission-blocking vaccine which induces antibodies that prevent establishment of a productive infection in mosquitos that feed on infected humans, thereby stopping the transmission cycle. One of the most promising targets for such a vaccine is the gamete surface protein, Pfs48/45. Here we establish a system for production of full-length Pfs48/45 and use this to raise a panel of monoclonal antibodies. We map the binding regions of these antibodies on Pfs48/45 and correlate the location of their epitopes with their transmission-blocking activity. Finally, we present the structure of the C-terminal domain of Pfs48/45 bound to the most potent transmission-blocking antibody, and provide key molecular information for future structure-guided immunogen design.
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http://dx.doi.org/10.1038/s41467-018-06340-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148045PMC
September 2018

A sporozoite-based vaccination platform against human malaria.

NPJ Vaccines 2018 24;3:33. Epub 2018 Aug 24.

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.

There is a pressing need for safe and highly effective () 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 () parasites as cross-species immunizing agents and as platforms for expression and delivery of CS (Vac). We show that both wild-type and Vac 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 hepatic but not blood infection, we show that Vac elicits cross-species cellular immune responses, as well as CS-specific antibodies that efficiently inhibit sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, Vac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development.
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http://dx.doi.org/10.1038/s41541-018-0068-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109154PMC
August 2018

Controlled Human Malaria Infection with Graded Numbers of NF135.C10- or NF166.C8-Infected Mosquitoes.

Am J Trop Med Hyg 2018 09 12;99(3):709-712. Epub 2018 Jul 12.

Department of Medical Microbiology, Radboud University Medical Center, Nijmegen, The Netherlands.

Controlled human malaria infections (CHMIs) with () parasites are well established. Exposure to five (NF54)-infected mosquitoes results in 100% infection rates in malaria-naïve volunteers. Recently clones NF135.C10 and NF166.C8 were generated for application in CHMIs. Here, we tested the clinical infection rates of these clones, using graded numbers of -infected mosquitoes. In a double-blind randomized trial, we exposed 24 malaria-naïve volunteers to bites from one, two, or five mosquitoes infected with NF135.C10 or NF166.C8. The primary endpoint was parasitemia by quantitative polymerase chain reaction. For both strains, bites by five infected mosquitoes resulted in parasitemia in 4/4 volunteers; 3/4 volunteers developed parasitemia after exposure to one or two infected mosquitoes infected with either clone. The prepatent period was 7.25 ± 4.0 days (median ± range). There were no serious adverse events and comparable clinical symptoms between all groups. These data confirm the eligibility of NF135.C10 and NF166.C8 for use in CHMI studies.
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http://dx.doi.org/10.4269/ajtmh.18-0194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169176PMC
September 2018