Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine.

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).

Infectivity of Plasmodium falciparum in Malaria-Naive Individuals Is Related to Knob Expression and Cytoadherence of the Parasite.

Plasmodium falciparum is the most virulent human malaria parasite because of its ability to cytoadhere in the microvasculature. Nonhuman primate studies demonstrated relationships among knob expression, cytoadherence, and infectivity. This has not been examined in humans. Cultured clinical-grade P. falciparum parasites (NF54, 7G8, and 3D7B) and ex vivo-derived cell banks were characterized. Knob and knob-associated histidine-rich protein expression, CD36 adhesion, and antibody recognition of parasitized erythrocytes (PEs) were evaluated. Parasites from the cell banks were administered to malaria-naive human volunteers to explore infectivity. For the NF54 and 3D7B cell banks, blood was collected from the study participants for in vitro characterization. All parasites were infective in vivo However, infectivity of NF54 was dramatically reduced. In vitro characterization revealed that unlike other cell bank parasites, NF54 PEs lacked knobs and did not cytoadhere. Recognition of NF54 PEs by immune sera was observed, suggesting P. falciparum erythrocyte membrane protein 1 expression. Subsequent recovery of knob expression and CD36-mediated adhesion were observed in PEs derived from participants infected with NF54. Knobless cell bank parasites have a dramatic reduction in infectivity and the ability to adhere to CD36. Subsequent infection of malaria-naive volunteers restored knob expression and CD36-mediated cytoadherence, thereby showing that the human environment can modulate virulence.

A repeat sequence domain of the ring-exported protein-1 of Plasmodium falciparum controls export machinery architecture and virulence protein trafficking.

The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.

A Plasmodium falciparum S33 proline aminopeptidase is associated with changes in erythrocyte deformability.

Infection with the apicomplexan parasite Plasmodium falciparum is a major cause of morbidity and mortality worldwide. One of the striking features of this parasite is its ability to remodel and decrease the deformability of host red blood cells, a process that contributes to disease. To further understand the virulence of Pf we investigated the biochemistry and function of a putative Pf S33 proline aminopeptidase (PfPAP). Unlike other P. falciparum aminopeptidases, PfPAP contains a predicted protein export element that is non-syntenic with other human infecting Plasmodium species. Characterization of PfPAP demonstrated that it is exported into the host red blood cell and that it is a prolyl aminopeptidase with a preference for N-terminal proline substrates. In addition genetic deletion of this exopeptidase was shown to lead to an increase in the deformability of parasite-infected red cells and in reduced adherence to the endothelial cell receptor CD36 under flow conditions. Our studies suggest that PfPAP plays a role in the rigidification and adhesion of infected red blood cells to endothelial surface receptors, a role that may make this protein a novel target for anti-disease interventions strategies.

Plasmodium falciparum neutral aminopeptidases: new targets for anti-malarials.

The neutral aminopeptidases M1 alanyl aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) of the human malaria parasite Plasmodium falciparum are targets for the development of novel anti-malarial drugs. Although the functions of these enzymes remain unknown, they are believed to act in the terminal stages of haemoglobin degradation, generating amino acids essential for parasite growth and development. Inhibitors of both enzymes are lethal to P. falciparum in culture and kill the murine malaria P. chabaudi in vivo. Recent biochemical, structural and functional studies provide the substrate specificity and mechanistic binding data needed to guide the development of more potent anti-malarial drugs. Together with biological studies, these data form the rationale for choosing PfM1AAP and PfM17LAP as targets for anti-malarial development.

Enhanced gametocyte formation in erythrocyte progenitor cells: a site-specific adaptation by Plasmodium falciparum.

Gametocytogenesis by Plasmodium falciparum is essential for transmission of the parasite from human to mosquito, yet developing gametocytes lack expression of surface proteins required for cytoadherence. Therefore, elimination from the circulation should occur unless they are sequestered in regions of low blood flow such as the extracellular spaces of the bone marrow. Our data indicate that gametocytogenesis is enhanced in the presence of erythroid progenitors found within the bone marrow. Furthermore, atomic force microscopy indicates that developing gametocytes undergo remarkable shifts in their erythrocyte membrane elasticity, which may allow them to be retained within the bone marrow until maturation.

Experimentally induced blood-stage Plasmodium vivax infection in healthy volunteers.

BACKGROUND: Major impediments to development of vaccines and drugs for Plasmodium vivax malaria are the inability to culture this species and the extreme difficulty in undertaking clinical research by experimental infection. METHODS: A parasite bank was collected from a 49-year-old woman with P. vivax infection, characterized, and used in an experimental infection study. RESULTS: The donor made a full recovery from malaria after collection of a parasite bank, which tested negative for agents screened for in blood donations. DNA sequence analysis of the isolate indicated that it was clonal. Two subjects inoculated with the isolate became polymerase chain reaction positive on days 8 and 9, with onset of symptoms and positive blood smears on day 14, when they were treated with artemether-lumefantrine, with rapid clinical and parasitologic response. Transcripts of the parasite gene pvs25 that is expressed in gametocytes, the life cycle stage infectious to mosquitoes, were first detected on days 11 and 12. CONCLUSIONS: This experimental system results in in vivo parasite growth, probably infectious to mosquitoes. It offers the opportunity to undertake studies previously impossible in P. vivax that will facilitate a better understanding of the pathology of vivax malaria and development of antimalarial drugs and vaccines. Trial Registration. ANZCTR: 12612001096842.

Temporal evaluation of commitment to sexual development in Plasmodium falciparum.

BACKGROUND: The production of gametocytes is essential for transmission of malaria parasites from the mammalian host to the mosquito vector. However the process by which the asexual blood-stage parasite undergoes commitment to sexual development is not well understood. This process is known to be sensitive to environmental stimuli and it has been suggested that a G protein dependent system may mediate the switch, but there is little evidence that the Plasmodium falciparum genome encodes heterotrimeric G proteins. Previous studies have indicated that the malaria parasite can interact with endogenous erythrocyte G proteins, and other components of the cyclic nucleotide pathway have been identified in P. falciparum. Also, the polypeptide cholera toxin, which induces commitment to gametocytogenesis is known to catalyze the ADP-ribosylation of the α(s) class of heterotrimeric G protein α subunits in mammalian systems has been reported to detect a number of G(α) subunits in P. falciparum-infected red cells. METHODS: Cholera toxin and Mas 7 (a structural analogue of Mastoparan) were used to assess the role played by putative G protein signalling in the commitment process, both are reported to interact with different components of classical Gas and Gai/o signalling pathways. Their ability to induce gametocyte production in the transgenic P. falciparum line Pfs16-GFP was determined and downstream effects on the secondary messenger cAMP measured. RESULTS: Treatment of parasite cultures with either cholera toxin or MAS 7 resulted in increased gametocyte production, but only treatment with MAS 7 resulted in a significant increase in cAMP levels. This indicates that MAS 7 acts either directly or indirectly on the P. falciparum adenylyl cyclase. CONCLUSION: The observation that cholera toxin treatment did not affect cAMP levels indicates that while addition of cholera toxin does increase gametocytogenesis the method by which it induces increased commitment is not immediately obvious, except that is unlikely to be via heterotrimeric G proteins.

Plasmodium falciparum gametocytes: with a view to a kill.

Drugs that kill or inhibit the sexual stages of Plasmodium in order to prevent transmission are important components of malaria control programmes. Reducing gametocyte carriage is central to the control of Plasmodium falciparum transmission as infection can result in extended periods of gametocytaemia. Unfortunately the number of drugs with activity against gametocytes is limited. Primaquine is currently the only licensed drug with activity against the sexual stages of malaria parasites and its use is hampered by safety concerns. This shortcoming is likely the result of the technical challenges associated with gametocyte studies together with the focus of previous drug discovery campaigns on asexual parasite stages. However recent emphasis on malaria eradication has resulted in an upsurge of interest in identifying compounds with activity against gametocytes. This review examines the gametocytocidal properties of currently available drugs as well as those in the development pipeline and examines the prospects for discovery of new anti-gametocyte compounds.

Dual stage synthesis and crucial role of cytoadherence-linked asexual gene 9 in the surface expression of malaria parasite var proteins.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate the adherence of parasite-infected red blood cells (IRBCs) to various host receptors. A previous study has shown that the parasite protein, cytoadherence-linked asexual gene 9 (CLAG9), is also essential for IRBC adherence. However, how CLAG9 influences this process remains unknown. In this study, we show that CLAG9 interacts with VAR2CSA, a PfEMP1 that mediates IRBC adherence to chondroitin 4-sulfate in the placenta. Importantly, our results show that the adherent parasites synthesize CLAG9 at two stages--the early ring and late trophozoite stages. Localization studies revealed that a substantial level of CLAG9 is located mainly at or in close proximity of the IRBC membrane in association with VAR2CSA. Upon treatment of IRBCs with trypsin, a significant amount of CLAG9 (≈150 kDa) was converted into ≈142-kDa polypeptide. Together these data demonstrate that a considerable amount of CLAG9 is embedded in the IRBC membrane such that at least a portion of the polypeptide at either N or C terminus is exposed on the cell surface. In parasites lacking CLAG9, VAR2CSA failed to express on the IRBC surface and was located within the parasite. Based on these findings, we propose that CLAG9 plays a critical role in the trafficking of PfEMP1s onto the IRBC surface. These results have important implications for the development of therapeutics for cerebral, placental, and other cytoadherence-associated malaria illnesses.

Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases.

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.

Genetic ablation of a Maurer's cleft protein prevents assembly of the Plasmodium falciparum virulence complex.

The malaria parasite Plasmodium falciparum assembles knob structures underneath the erythrocyte membrane that help present the major virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1). Membranous structures called Maurer's clefts are established in the erythrocyte cytoplasm and function as sorting compartments for proteins en route to the RBC membrane, including the knob-associated histidine-rich protein (KAHRP), and PfEMP1. We have generated mutants in which the Maurer's cleft protein, the ring exported protein-1 (REX1) is truncated or deleted. Removal of the C-terminal domain of REX1 compromises Maurer's cleft architecture and PfEMP1-mediated cytoadherance but permits some trafficking of PfEMP1 to the erythrocyte surface. Deletion of the coiled-coil region of REX1 ablates PfEMP1 surface display, trapping PfEMP1 at the Maurer's clefts. Complementation of mutants with REX1 partly restores PfEMP1-mediated binding to the endothelial cell ligand, CD36. Deletion of the coiled-coil region or complete deletion of REX1 is tightly associated with the loss of a subtelomeric region of chromosome 2, encoding KAHRP and other proteins. A KAHRP-green fluorescent protein (GFP) fusion expressed in the REX1-deletion parasites shows defective trafficking. Thus, loss of functional REX1 directly or indirectly ablates the assembly of the P. falciparum virulence complex at the surface of host erythrocytes.

The aminopeptidase inhibitor CHR-2863 is an orally bioavailable inhibitor of murine malaria.

Malaria remains a significant risk in many areas of the world, with resistance to the current antimalarial pharmacopeia an ever-increasing problem. The M1 alanine aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are believed to play a role in the terminal stages of digestion of host hemoglobin and thereby generate a pool of free amino acids that are essential for parasite growth and development. Here, we show that an orally bioavailable aminopeptidase inhibitor, CHR-2863, is efficacious against murine malaria.

Anti-malarial drugs: how effective are they against Plasmodium falciparum gametocytes?

BACKGROUND: Recent renewed emphasis on the eradication of malaria has highlighted the need for more tools with which to achieve this ambitious goal. One high priority area is the need to determine the gametocytocidal activity of both currently used anti-malarial drugs and those in the development pipeline. However, testing the activity of compounds against Plasmodium falciparum gametocytes is technically challenging both in vivo and in vitro. METHODS: Here the use of a simple robust assay to screen a panel of currently used and experimental anti-malarial drugs against mature P. falciparum gametocytes is described. RESULTS: Eight of 44 compounds tested reduced gametocyte viability by at least 50% and three showed IC50 values in nM range. CONCLUSIONS: There is a need to identify new compounds with activity against late stage gametocytes and the information provided by this in vitro assay is a valuable first step, which can guide future clinical studies.

Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase.

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH(2)]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.

BACKGROUND: Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. METHODS: Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. RESULTS: The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. CONCLUSIONS: This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue.

Targeting of the ring exported protein 1 to the Maurer's clefts is mediated by a two-phase process.

Early development of Plasmodium falciparum within the erythrocyte is characterized by the large-scale export of proteins to the host cell. In many cases, export is mediated by a short sequence called the Plasmodium export element (PEXEL) or vacuolar transport signal; however, a number of previously characterized exported proteins do not contain such an element. In this study, we investigated the mechanisms of export of the PEXEL-negative ring exported protein 1 (REX1). This protein localizes to the Maurer's clefts, parasite-induced structures in the host-cell cytosol. Transgenic parasites expressing green fluorescent protein-REX1 chimeras revealed that the single hydrophobic stretch plus an additional 10 amino acids mediate the export of REX1. Biochemical characterization of these chimeras indicated that REX1 was exported as a soluble protein. Inclusion of a sequence containing a predicted coiled-coil motif led to the correct localization of REX1 at the Maurer's clefts, suggesting that association with the clefts occurs at the final stage of protein export only. These results indicate that PEXEL-negative exported proteins can be exported in a soluble state and that sequences without any apparent resemblance to a PEXEL motif can mediate export across the parasitophorous vacuole membrane.

Antimalarial asexual stage-specific and gametocytocidal activities of HIV protease inhibitors.

The stage-specific antimalarial activities of a panel of antiretroviral protease inhibitors (PIs), including two nonpeptidic PIs (tipranavir and darunavir), were tested in vitro against Plasmodium falciparum. While darunavir demonstrated limited antimalarial activity (effective concentration [EC(50)], >50 microM), tipranavir was active at clinically relevant concentrations (EC(50), 12 to 21 microM). Saquinavir, lopinavir, and tipranavir preferentially inhibited the growth of mature asexual-stage parasites (24 h postinvasion). While all of the PIs tested inhibited gametocytogenesis, tipranavir was the only one to exhibit gametocytocidal activity.

Effect of antimalarial drugs on Plasmodium falciparum gametocytes.

Gametocytes are the sexual stage of the malaria parasite and are essential for transmission to the mosquito. Antimalarial drugs have been reported to affect gametocyte production in vivo, which leads to a potential increase in transmission. We used transgenic Plasmodium falciparum parasites expressing a green fluorescent protein tag in a fluorescence-activated cell sorting-based assay to measure the effect of 8 antimalarial drugs on gametocyte production in vitro. Exposure to antimalarial drugs resulted in an increase in the number of gametocytes in test cultures. Although a dose-dependent reduction in late-stage gametocyte viability was observed, none of the drugs tested statistically significantly reduced gametocyte numbers.

Anti-Giardia Drug Discovery: Current Status and Gut Feelings.

Giardia parasites are ubiquitous protozoans of global importance that impact a wide range of animals including humans. They are the most common enteric pathogen of cats and dogs in developed countries and infect ∼1 billion people worldwide. While Giardia infections can be asymptomatic, they often result in severe and chronic diseases. There is also mounting evidence that they are linked to postinfection disorders. Despite growing evidence of the widespread morbidity associated with Giardia infections, current treatment options are limited to compound classes with broad antimicrobial activity. Frontline anti-Giardia drugs are also associated with increasing drug resistance and treatment failures. To improve the health and well-being of millions, new selective anti-Giardia drugs are needed alongside improved health education initiatives. Here we discuss current treatment options together with recent advances and gaps in drug discovery. We also propose criteria to guide the discovery of new anti-Giardia compounds.