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).

Developing a coding taxonomy to analyze dental regulatory complaints.

BACKGROUND: As part of their mandate to protect the public, dental regulatory authorities (DRA) in Canada are responsible for investigating complaints made by members of the public. To gain an understanding of the nature of and trends in complaints made to the Royal College of Dental Surgeons of Ontario (RCDSO), Canada's largest DRA, a coding taxonomy was developed for systematic analysis of complaints. METHODS: The taxonomy was developed through a two-pronged approach. First, the research team searched for existing complaints frameworks and integrated data from a variety of sources to ensure applicability to the dental context in terms of the generated items/complaint codes in the taxonomy. Second, an anonymized sample of complaint letters made by the public to the RCDSO (n = 174) were used to refine the taxonomy. This sample was further used to assess the feasibility of use in a larger content analysis of complaints. Inter-coder reliability was also assessed using a separate sample of letters (n = 110). RESULTS: The resulting taxonomy comprised three domains (Clinical Care and Treatment, Management and Access, and Relationships and Conduct), with seven categories, 23 sub-categories, and over 100 complaint codes. Pilot testing for the feasibility and applicability of the taxonomy's use for a systematic analysis of complaints proved successful. CONCLUSIONS: The resulting coding taxonomy allows for reliable documentation and interpretation of complaints made to a DRA in Canada and potentially other jurisdictions, such that the nature of and trends in complaints can be identified, monitored and used in quality assurance and improvement.

A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial.

BACKGROUND: Although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)-compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials. METHODS: Genetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product. RESULTS: Two hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). CONCLUSION: Large-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.

Metabolomics-Based Elucidation of Active Metabolic Pathways in Erythrocytes and HSC-Derived Reticulocytes.

A detailed analysis of the metabolic state of human-stem-cell-derived erythrocytes allowed us to characterize the existence of active metabolic pathways in younger reticulocytes and compare them to mature erythrocytes. Using high-resolution LC-MS-based untargeted metabolomics, we found that reticulocytes had a comparatively much richer repertoire of metabolites, which spanned a range of metabolite classes. An untargeted metabolomics analysis using stable-isotope-labeled glucose showed that only glycolysis and the pentose phosphate pathway actively contributed to the biosynthesis of metabolites in erythrocytes, and these pathways were upregulated in reticulocytes. Most metabolite species found to be enriched in reticulocytes were residual pools of metabolites produced by earlier erythropoietic processes, and their systematic depletion in mature erythrocytes aligns with the simplification process, which is also seen at the cellular and the structural level. Our work shows that high-resolution LC-MS-based untargeted metabolomics provides a global coverage of the biochemical species that are present in erythrocytes. However, the incorporation of stable isotope labeling provides a more accurate description of the active metabolic processes that occur in each developmental stage. To our knowledge, this is the first detailed characterization of the active metabolic pathways of the erythroid lineage, and it provides a rich database for understanding the physiology of the maturation of reticulocytes into mature erythrocytes.

Host reticulocytes provide metabolic reservoirs that can be exploited by malaria parasites.

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5'-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites.

Immune responses during cutaneous and visceral leishmaniasis.

SUMMARY Leishmania are protozoan parasites spread by a sandfly insect vector and causing a spectrum of diseases collectively known as leishmaniasis. The disease is a significant health problem in many parts of the world, resulting in an estimated 1·3 million new cases and 30 000 deaths annually. Current treatment is based on chemotherapy, which is difficult to administer, expensive and becoming ineffective in several endemic regions. To date there is no vaccine against leishmaniasis, although extensive evidence from studies in animal models indicates that solid protection can be achieved upon immunization. This review focuses on immune responses to Leishmania in both cutaneous and visceral forms of the disease, pointing to the complexity of the immune response and to a range of evasive mechanisms utilized by the parasite to bypass those responses. The amalgam of innate and acquired immunity combined with the paucity of data on the human immune response is one of the major problems currently hampering vaccine development and implementation.

NK cells and conventional dendritic cells engage in reciprocal activation for the induction of inflammatory responses during Plasmodium berghei ANKA infection.

Cerebral malaria (CM) is the most severe syndrome associated with Plasmodium falciparum infections. Experimental evidence suggests that disease results from the sequestration of parasitized-red blood cells (pRBCs) together with inflammatory leukocytes within brain capillaries. We have previously shown that NK cells stimulate migration of CXCR3(+) T cells to the brain of Plasmodium berghei ANKA-infected mice. Here we investigated whether interactions between NK cells and dendritic cells (DCs) are required for the induction of T cell responses involved in disease. For that, NK cell-depleted and control mice were infected with transgenic parasites expressing model T cell epitopes. T cells from TCR transgenic mice specific for those epitopes were adoptively transferred and proliferation was determined. NK cell depletion significantly reduced CD8(+) but not CD4(+) DC-mediated T cell priming. Lack of NK cells did not compromise CD8(+) T cell responses in IL-12(-/-) mice, suggesting that NK cells stimulate IL-12 output by DCs required for optimal T cell priming. The contribution of DCs to NK cell function was also investigated. DC depletion and genetic deletion of IL-12 dramatically reduced NK cell-mediated IFN-γ responses to malaria. Thus NK cells and DCs engage in reciprocal activation for the induction of inflammatory responses involved in severe malaria.

Functional analysis of Leishmania cyclopropane fatty acid synthetase.

The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.

Novel indoleamine 2,3-dioxygenase-1 inhibitors from a multistep in silico screen.

Indoleamine 2,3-dioxygenase-1 (IDO-1) is a heme containing enzyme that catalyses the initial step in the major pathway of l-tryptophan catabolism; the kynurenine pathway. A large body of evidence has been accumulating for its immunosuppressive and tumoural escape roles and its applicability as a therapeutic target. Of particular interest is the possibility that IDO-1 inhibition may arrest, and sometimes revert, tumour growth. There exists a continuing need for the development of new and specific inhibitors for IDO-1, and we have created three pharmacophores designed to aid in this search. Initial database hits were further screened using Kier flexibility and a 'What-If' docking technique, designed to overcome the inherent limitations of today's forcefields with regards to heme chemistry. Eighteen compounds were tested in vitro, yielding four novel inhibitors with low micromolar IC(50) values, comparable with current inhibitors.

Development of Vaccines against Visceral Leishmaniasis.

Leishmaniasis is a neglected disease resulting in a global morbidity of 2,090 thousand Disability-Adjusted Life Years and a mortality rate of approximately 60,000 per year. Among the three clinical forms of leishmaniasis (cutaneous, mucosal, and visceral), visceral leishmaniasis (VL) accounts for the majority of mortality, as if left untreated VL is almost always fatal. Caused by infection with Leishmania donovani or L. infantum, VL represents a serious public health problem in endemic regions and is rapidly emerging as an opportunistic infection in HIV patients. To date, no vaccine exists for VL or any other form of leishmaniasis. In endemic areas, the majority of those infected do not develop clinical symptoms and past infection leads to robust immunity against reinfection. Thus the development of vaccine for Leishmania is a realistic public health goal, and this paper summarizes advances in vaccination strategies against VL.

Comparative expression profiling of Leishmania: modulation in gene expression between species and in different host genetic backgrounds.

BACKGROUND: Genome sequencing of Leishmania species that give rise to a range of disease phenotypes in the host has revealed highly conserved gene content and synteny across the genus. Only a small number of genes are differentially distributed between the three species sequenced to date, L. major, L. infantum and L. braziliensis. It is not yet known how many of these genes are expressed in the disease-promoting intracellular amastigotes of these species or whether genes conserved between the species are differentially expressed in the host. METHODS/PRINCIPAL FINDINGS: We have used customised oligonucleotide microarrays to confirm that all of the differentially distributed genes identified by genome comparisons are expressed in intracellular amastigotes, with only a few of these subject to regulation at the RNA level. In the first large-scale study of gene expression in L. braziliensis, we show that only approximately 9% of the genes analysed are regulated in their RNA expression during the L. braziliensis life cycle, a figure consistent with that observed in other Leishmania species. Comparing amastigote gene expression profiles between species confirms the proposal that Leishmania transcriptomes undergo little regulation but also identifies conserved genes that are regulated differently between species in the host. We have also investigated whether host immune competence influences parasite gene expression, by comparing RNA expression profiles in L. major amastigotes derived from either wild-type (BALB/c) or immunologically compromised (Rag2(-/-) gamma(c) (-/-)) mice. While parasite dissemination from the site of infection is enhanced in the Rag2(-/-) gamma(c) (-/-) genetic background, parasite RNA expression profiles are unperturbed. CONCLUSION/SIGNIFICANCE: These findings support the hypothesis that Leishmania amastigotes are pre-adapted for intracellular survival and undergo little dynamic modulation of gene expression at the RNA level. Species-specific parasite factors contributing to virulence and pathogenicity in the host may be limited to the products of a small number of differentially distributed genes or the differential regulation of conserved genes, either of which are subject to translational and/or post-translational controls.

Preferential invasion of reticulocytes during late-stage Plasmodium berghei infection accounts for reduced circulating reticulocyte levels.

Insufficient circulating reticulocytes have been observed during severe malarial anaemia in both human and murine infection, and are often attributed to reduced production of red cell precursors. However, a number of Plasmodium species display a preference for invading reticulocytes rather than erythrocytes. Thus, the reduction in circulating reticulocyte numbers may arise as a result both of increased parasitization and lysis of reticulocytes, as well as decreased production. We have analysed both circulating reticulocyte numbers and the percentage of infected reticulocytes during murine Plasmodium berghei infection. We found a large reduction in circulating numbers when compared with an equivalent chemically induced anaemia. However, mathematical analysis of parasite and red cell numbers revealed the preference of P. berghei for reticulocytes to be approximately 150-fold over that for erythrocytes, leading to increased destruction of reticulocytes. Although erythropoietic suppression is evident during the first week of P. berghei infection, this preferential infection and destruction of reticulocytes is sufficient to mediate ongoing reduced levels of circulating reticulocytes during the latter stages of infection, following compensatory erythropoiesis in response to haemolytic anaemia.

Regulation of surface coat exchange by differentiating African trypanosomes.

African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

Severe malarial anemia of low parasite burden in rodent models results from accelerated clearance of uninfected erythrocytes.

Severe malarial anemia (SMA) is the most frequent life-threatening complication of malaria and may contribute to the majority of malarial deaths worldwide. To explore the mechanisms of pathogenesis, we developed a novel murine model of SMA in which parasitemias peaked around 1.0% of circulating red blood cells (RBCs) and yet hemoglobin levels fell to 47% to 56% of baseline. The severity of anemia was independent of the level of peak or cumulative parasitemia, but was linked kinetically to the duration of patent infection. In vivo biotinylation analysis of the circulating blood compartment revealed that anemia arose from accelerated RBC turnover. Labeled RBCs were reduced to 1% of circulating cells by 8 days after labeling, indicating that the entire blood compartment had been turned over in approximately one week. The survival rate of freshly transfused RBCs was also markedly reduced in SMA animals, but was not altered when RBCs from SMA donors were transferred into naive recipients, suggesting few functional modifications to target RBCs. Anemia was significantly alleviated by depletion of either phagocytic cells or CD4+ T lymphocytes. This study demonstrates that immunologic mechanisms may contribute to SMA by promoting the accelerated turnover of uninfected RBCs.

The natural killer complex regulates severe malarial pathogenesis and influences acquired immune responses to Plasmodium berghei ANKA.

The natural killer complex (NKC) is a genetic region of highly linked genes encoding several receptors involved in the control of NK cell function. The NKC is highly polymorphic, and allelic variability of various NKC loci has been demonstrated in inbred mice. Making use of BALB.B6-Cmv1r congenic mice, in which the NKC from disease-susceptible C57BL/6 mice has been introduced into the disease-resistant BALB/c background, we show here that during murine malaria infection, the NKC regulates a range of pathophysiological syndromes such as cerebral malaria, pulmonary edema, and severe anemia, which contribute to morbidity and mortality in human malaria. Parasitemia levels were not affected by the NKC genotype, indicating that control of malarial fatalities by the NKC cells does not operate through effects on parasite growth rate. Parasite-specific antibody responses and the proinflammatory gene transcription profile, as well as the TH1/TH2 balance, also appeared to be influenced by NKC genotype, providing evidence that this region, known to control innate immune responses via NK and/or NK T-cell activation, can also significantly regulate acquired immunity to infection. To date, NKC-encoded innate system receptors have been shown mainly to regulate viral infections. Our data provide evidence for critical NKC involvement in the broad immunological responses to a protozoan parasite.

Ligand self-recognition in the stereoselective assembly of [2 + 2] metallomacrocycles from racemic chiral bisbipyridyl molecular clefts and zinc(II).

The synthesis of racemic and optically pure ligand L, in which two 6,6'-disubstituted bipyridines are connected by methyleneoxy linkers to the molecular cleft dibenzobicyclo[b,f][3.3.1]nona-5a,6a-diene-6,12-dione, is reported. In the presence of 2 equivalents of zinc(ii) trifluoromethansulfonate (+/-)- undergoes slow reversible coordination over 24 h to form a pair of enantiomeric [2 + 2] metallomacrocycles, [Zn2(+)L2](OTf)(4) and [Zn2(-)L2](OTf)(4) respectively, that contain either two (+)-L ligands or two (-)-L ligands. This assignment was confirmed by independent studies with either (+)-L or (-)-L which formed the same complexes but at a significantly faster rate (3 h), and circular dichroism spectra of [Zn2(+)L2](OTf)(4) and [Zn2(-)L2](OTf)(4) which gave signals of the same intensity with the opposite sign. Treatment of (+/-)-L or optically pure L with copper(I) showed rapid formation of a mixture of oligomers as well as the [2 + 2] metallomacrocycle. The complex Zn2L2(OTf)(4) exhibits slow exchange between two species on the NMR time scale at room temperature. The results are consistent with the formation of a library of metal complexes in which the zinc(ii) binds initially to the most accessible bipyridyl binding sites in (+/-)-. Equilibration over several hours results in self-recognition of enantiomeric ligands to form a pair of enantiomeric metallomacrocycles, which have been tentatively assigned as having the helical configuration. Slow exchange is attributed to the preference for both metal centres to adopt 6-coordinate geometries involving the linker oxygens, but are limited to exchanging 5-coordinate complexes due to the shape of the cleft and the short linker.

Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria.

The malaria parasite Plasmodium falciparum infects 5-10% of the world's population and kills two million people annually. Fatalities are thought to result in part from pathological reactions initiated by a malarial toxin. Glycosylphosphatidylinositol (GPI) originating from the parasite has the properties predicted of a toxin; however, a requirement for toxins in general and GPI in particular in malarial pathogenesis and fatality remains unproven. As anti-toxic vaccines can be highly effective public health tools, we sought to determine whether anti-GPI vaccination could prevent pathology and fatalities in the Plasmodium berghei/rodent model of severe malaria. The P. falciparum GPI glycan of the sequence NH(2)-CH(2)-CH(2)-PO(4)-(Man alpha 1-2)6Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcNH(2)alpha 1-6myo-inositol-1,2-cyclic-phosphate was chemically synthesized, conjugated to carriers, and used to immunize mice. Recipients were substantially protected against malarial acidosis, pulmonary oedema, cerebral syndrome and fatality. Anti-GPI antibodies neutralized pro-inflammatory activity by P. falciparum in vitro. Thus, we show that GPI is a significant pro-inflammatory endotoxin of parasitic origin, and that several disease parameters in malarious mice are toxin-dependent. GPI may contribute to pathogenesis and fatalities in humans. Synthetic GPI is therefore a prototype carbohydrate anti-toxic vaccine against malaria.