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There is a pressing need for safe and highly effective Plasmodium falciparum ( Pf ) malaria vaccines. The circumsporozoite protein (CS), expressed on sporozoites and during early hepatic stages, is a leading target vaccine candidate, but clinical efficacy has been modest so far. Conversely, whole-sporozoite (WSp) vaccines have consistently shown high levels of sterilizing immunity and constitute a promising approach to effective immunization against malaria. Here, we describe a novel WSp malaria vaccine that employs transgenic sporozoites of rodent P. berghei ( Pb ) parasites as cross-species immunizing agents and as platforms for expression and delivery of Pf CS ( Pb Vac). We show that both wild-type Pb and Pb 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 Pb hepatic but not blood infection, we show that Pb Vac elicits cross-species cellular immune responses, as well as Pf CS-specific antibodies that efficiently inhibit Pf sporozoite liver invasion in human hepatocytes and in mice with humanized livers. Thus, Pb Vac is safe and induces functional immune responses in preclinical studies, warranting clinical testing and development., Competing Interests: A.M.M., M.M.M., and M.P. are inventors on a patent or patent application issued, allowed or filed internationally, covering parts of this work. All other authors declare no competing interests.

Vivax malaria remains one of the most serious and neglected tropical diseases, with 132 to 391 million clinical cases per year and 2.5 billion people at risk of infection. A vaccine against Plasmodium vivax could have more impact than any other intervention, and the use of a vaccine targeting multiple antigens may result in higher efficacy against sporozoite infection than targeting a single antigen. Here, two leading P. vivax preerythrocytic vaccine candidate antigens, the P. vivax circumsporozoite protein (PvCSP) and the thrombospondin-related adhesion protein (PvTRAP) were delivered as a combined vaccine. This strategy provided a dose-sparing effect, with 100% sterile protection in mice using doses that individually conferred low or no protection, as with the unadjuvanted antigens PvTRAP (0%) and PvCSP (50%), and reached protection similar to that of adjuvanted components. Efficacy against malaria infection was assessed using a new mouse challenge model consisting of a double-transgenic Plasmodium berghei parasite simultaneously expressing PvCSP and PvTRAP used in mice immunized with the virus-like particle (VLP) Rv21 previously reported to induce high efficacy in mice using Matrix-M adjuvant, while PvTRAP was concomitantly administered in chimpanzee adenovirus and modified vaccinia virus Ankara (MVA) vectors (viral-vectored TRAP, or vvTRAP) to support effective induction of T cells. We examined immunity elicited by these vaccines in the context of two adjuvants approved for human use (AddaVax and Matrix-M). Matrix-M supported the highest anti-PvCSP antibody titers when combined with Rv21, and, interestingly, mixing PvCSP Rv21 and PvTRAP viral vectors enhanced immunity to malaria over levels provided by single vaccines., (Copyright © 2018 Atcheson et al.)

Protection against a malaria infection can be achieved by immunization with live-attenuated Plasmodium sporozoites and while the precise mechanisms of protection remain unknown, T cell responses are thought to be critical in the elimination of infected liver cells. In cancer immunotherapies, agonistic antibodies that target T cell surface proteins, such as CD27, OX40 (CD134), and 4-1BB (CD137), have been used to enhance T cell function by increasing co-stimulation. In this study, we have analyzed the effect of agonistic OX40 monoclonal antibody treatment on protective immunity induced in mice immunized with genetically attenuated parasites (GAPs). OX40 stimulation enhanced protective immunity after vaccination as shown by an increase in the number of protected mice and delay to blood-stage infection after challenge with wild-type sporozoites. Consistent with the enhanced protective immunity enforced OX40 stimulation resulted in an increased expansion of antigen-experienced effector (CD11a hi CD44 hi ) CD8 + and CD4 + T cells in the liver and spleen and also increased IFN-γ and TNF producing CD4 + T cells in the liver and spleen. In addition, GAP immunization plus α-OX40 treatment significantly increased sporozoite-specific IgG responses. Thus, we demonstrate that targeting T cell costimulatory receptors can improve sporozoite-based vaccine efficacy.

With the increasing prevalence of artemisinin-resistant malaria parasites, a highly efficacious and durable vaccine for malaria is urgently required. We have developed an experimental virus-vectored vaccine platform based on an envelope-modified baculovirus dual-expression system (emBDES). Here, we show a conceptually new vaccine platform based on an adenovirus-prime/emBDES-boost heterologous immunization regimen expressing the Plasmodium falciparum circumsporozoite protein (PfCSP). A human adenovirus 5-prime/emBDES-boost heterologous immunization regimen consistently achieved higher sterile protection against transgenic P. berghei sporozoites expressing PfCSP after a mosquito-bite challenge than reverse-ordered or homologous immunization. This high protective efficacy was also achieved with a chimpanzee adenovirus 63-prime/emBDES-boost heterologous immunization regimen against an intravenous sporozoite challenge. Thus, we show that the adenovirus-prime/emBDES-boost heterologous immunization regimen confers sterile protection against sporozoite challenge by two individual routes, providing a promising new malaria vaccine platform for future clinical use.

Plasmodium gametocytes are the sexual forms of the malaria parasite essential for transmission to mosquitoes. To better understand how gametocytes differ from asexual blood-stage parasites, we performed a systematic analysis of available 'omics data for P. falciparum and other Plasmodium species. 18 transcriptomic and proteomic data sets were evaluated for the presence of curated "gold standards" of 41 gametocyte-specific versus 46 non-gametocyte genes and integrated using Bayesian probabilities, resulting in gametocyte-specificity scores for all P. falciparum genes. To illustrate the utility of the gametocyte score, we explored newly predicted gametocyte-specific genes as potential biomarkers of gametocyte carriage and exposure. We analyzed the humoral immune response in field samples against 30 novel gametocyte-specific antigens and found five antigens to be differentially recognized by gametocyte carriers as compared to malaria-infected individuals without detectable gametocytes. We also validated the gametocyte-specificity of 15 identified gametocyte transcripts on culture material and samples from naturally infected individuals, resulting in eight transcripts that were >1000-fold higher expressed in gametocytes compared to asexual parasites and whose transcript abundance allowed gametocyte detection in naturally infected individuals. Our integrated genome-wide gametocyte-specificity scores provide a comprehensive resource to identify targets and monitor P. falciparum gametocytemia.

A large research effort is currently underway to find an effective and affordable malaria vaccine. Tools that enable the rapid evaluation of protective immune responses are essential to vaccine development as they can provide selection criteria to rank order vaccine candidates. In this study we have revisited the Inhibition of Sporozoite Invasion (ISI) assay to assess the ability of antibodies to inhibit sporozoite infection of hepatocytes. By using GFP expressing sporozoites of the rodent parasite P. berghei we are able to robustly quantify parasite infection of hepatocyte cell lines by flow cytometry. In conjunction with recently produced transgenic P. berghei parasites that express P. falciparum sporozoite antigens, we have been able to use this assay to measure antibody mediated inhibition of sporozoite invasion against one of the lead malaria antigens P. falciparum CSP. By combining chimeric rodent parasites expressing P. falciparum antigens and a flow cytometric readout of infection, we are able to robustly assess vaccine-induced antibodies, from mice, rhesus macaques and human clinical trials, for their functional ability to block sporozoite invasion of hepatocytes.

In humans and murine models of malaria, intradermal immunization (ID-I) with genetically attenuated sporozoites that arrest in liver induces lower protective immunity than intravenous immunization (IV-I). It is unclear whether this difference is caused by fewer sporozoites migrating into the liver or by suboptimal hepatic and injection site-dependent immune responses. We therefore developed a Plasmodium yoelii immunization/boost/challenge model to examine parasite liver loads as well as hepatic and lymph node immune responses in protected and unprotected ID-I and IV-I animals. Despite introducing the same numbers of genetically attenuated parasites in the liver, ID-I resulted in lower sterile protection (53-68%) than IV-I (93-95%). Unprotected mice developed less sporozoite-specific CD8 + and CD4 + effector T-cell responses than protected mice. After immunization, ID-I mice showed more interleukin-10-producing B and T cells in livers and skin-draining lymph nodes, but fewer hepatic CD8 memory T cells and CD8 + dendritic cells compared to IV-I mice. Our results indicate that the lower protection efficacy obtained by intradermal sporozoite administration is not linked to low hepatic parasite numbers as presumed before, but correlates with a shift towards regulatory immune responses. Overcoming these immune suppressive responses is important not only for live-attenuated malaria vaccines but also for other live vaccines administered in the skin.

Introduction: Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.

Although malaria is a life-threatening disease with severe complications, most people develop partial immunity and suffer from mild symptoms. However, incomplete recovery from infection causes chronic illness, and little is known of the potential outcomes of this chronicity. We found that malaria causes bone loss and growth retardation as a result of chronic bone inflammation induced by Plasmodium products. Acute malaria infection severely suppresses bone homeostasis, but sustained accumulation of Plasmodium products in the bone marrow niche induces MyD88-dependent inflammatory responses in osteoclast and osteoblast precursors, leading to increased RANKL expression and overstimulation of osteoclastogenesis, favoring bone resorption. Infection with a mutant parasite with impaired hemoglobin digestion that produces little hemozoin, a major Plasmodium by-product, did not cause bone loss. Supplementation of alfacalcidol, a vitamin D3 analog, could prevent the bone loss. These results highlight the risk of bone loss in malaria-infected patients and the potential benefits of coupling bone therapy with antimalarial treatment., (Copyright © 2017, American Association for the Advancement of Science.)

Four different vaccine platforms, each targeting the human malaria parasite Plasmodium vivax cell-traversal protein for ookinetes and sporozoites ( Pv CelTOS), were generated and assessed for protective efficacy. These platforms consisted of a recombinant chimpanzee adenoviral vector 63 (ChAd63) expressing Pv CelTOS (Ad), a recombinant modified vaccinia virus Ankara expressing Pv CelTOS (MVA), Pv CelTOS conjugated to bacteriophage Qβ virus-like particles (VLPs), and a recombinant Pv CelTOS protein expressed in eukaryotic HEK293T cells (protein). Inbred BALB/c mice and outbred CD-1 mice were immunized using the following prime-boost regimens: Ad-MVA, Ad-VLPs, and Ad-protein. Protective efficacy against sporozoite challenge was assessed after immunization using a novel chimeric rodent Plasmodium berghei parasite ( Pb-Pv CelTOS). This chimeric parasite expresses P. vivax CelTOS in place of the endogenous P. berghei CelTOS and produces fully infectious sporozoites. A single Ad immunization in BALB/c and CD-1 mice induced anti- Pv CelTOS antibodies which were boosted efficiently using MVA, VLP, or protein immunization. Pv CelTOS-specific gamma interferon- and tumor necrosis factor alpha-producing CD8 + T cells were induced at high frequencies by all prime-boost regimens in BALB/c mice but not in CD-1 mice; in CD-1 mice, they were only marginally increased after boosting with MVA. Despite the induction of anti- Pv CelTOS antibodies and Pv CelTOS-specific CD8 + T-cell responses, only low levels of protective efficacy against challenge with Pb-Pv CelTOS sporozoites were obtained using any immunization strategy. In BALB/c mice, no immunization regimens provided significant protection against a Pb-Pv CelTOS chimeric sporozoite challenge. In CD-1 mice, modest protective efficacy against challenge with chimeric P. berghei sporozoites expressing either Pv CelTOS or P. falciparum CelTOS was observed using the Ad-protein vaccination regimen., (Copyright © 2017 Alves et al.)

Efforts are under way to improve the efficacy of subunit malaria vaccines through assessments of new adjuvants, vaccination platforms, and antigens. In this study, we further assessed the Plasmodium falciparum antigen upregulated in infective sporozoites 3 (PfUIS3) as a vaccine candidate. PfUIS3 was expressed in the viral vectors chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) and used to immunize mice in a prime-boost regimen. We previously demonstrated that this regimen could provide partial protection against challenge with chimeric P. berghei parasites expressing PfUIS3. We now show that ChAd63-MVA PfUIS3 can also provide partial cross-species protection against challenge with wild-type P. berghei parasites. We also show that PfUIS3-specific cellular memory responses could be recalled in human volunteers exposed to P. falciparum parasites in a controlled human malaria infection study. When ChAd63-MVA PfUIS3 was coadministered with the vaccine candidate P. falciparum thrombospondin-related adhesion protein (PfTRAP) expressed in the ChAd63-MVA system, there was no significant change in immunogenicity to either vaccine. However, when mice were challenged with double chimeric P. berghei - P. falciparum parasites expressing both PfUIS3 and PfTRAP, vaccine efficacy was improved to 100% sterile protection. This synergistic effect was evident only when the two vaccines were mixed and administered at the same site. We have therefore demonstrated that vaccination with PfUIS3 can induce a consistent delay in patent parasitemia across mouse strains and against chimeric parasites expressing PfUIS3 as well as wild-type P. berghei ; when this vaccine is combined with another partially protective regimen (ChAd63-MVA PfTRAP), complete protection is induced., (Copyright © 2017 Longley et al.)

Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission., (Copyright © 2017 Espinosa et al.)

Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes., (© 2015 Lin et al.)

Sexual reproduction is an obligate step in the life cycle of many parasites, including the causative agents of malaria (Plasmodium). Mixed-species infections are common in nature and consequently, interactions between heterospecific gametes occur. Given the importance of managing gene flow across parasite populations, remarkably little is understood about how reproductive isolation between species is maintained. We use the rodent malaria parasites P. berghei and P. yoelii to investigate the ecology of mixed-species mating groups, identify proteins involved in pre-zygotic barriers, and examine their evolution. Specifically, we show that (i) hybridization occurs, but at low frequency; (ii) hybridization reaches high levels when female gametes lack the surface proteins P230 or P48/45, demonstrating that these proteins are key for pre-zygotic reproductive isolation; (iii) asymmetric reproductive interference occurs, where the fertility of P. berghei gametes is reduced in the presence of P. yoelii and (iv) as expected for gamete recognition proteins, strong positive selection acts on a region of P230 and P47 (P48/45 paralogue). P230 and P48/45 are leading candidates for interventions to block malaria transmission. Our results suggest that depending on the viability of hybrids, applying such interventions to populations where mixed-species infections occur could either facilitate or hinder malaria control.

We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.

A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

Model antigens are frequently introduced into pathogens to study determinants that influence T-cell responses to infections. To address whether an antigen's subcellular location influences the nature and magnitude of antigen-specific T-cell responses, we generated Plasmodium berghei parasites expressing the model antigen ovalbumin (OVA) either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). For cytosolic expression, OVA alone or conjugated to mCherry was expressed from a strong constitutive promoter (OVAhsp70 or OVA::mCherryhsp70); for PVM expression, OVA was fused to HEP17/EXP1 (OVA::Hep17hep17). Unexpectedly, OVA expression in OVAhsp70 parasites was very low, but when OVA was fused to mCherry (OVA::mCherryhsp70), it was highly expressed. OVA expression in OVA::Hep17hep17 parasites was strong but significantly less than that in OVA::mCherryhsp70 parasites. These transgenic parasites were used to examine the effects of antigen subcellular location and expression level on the development of T-cell responses during blood-stage infections. While all OVA-expressing parasites induced activation and proliferation of OVA-specific CD8(+) T cells (OT-I) and CD4(+) T cells (OT-II), the level of activation varied: OVA::Hep17hep17 parasites induced significantly stronger splenic and intracerebral OT-I and OT-II responses than those of OVA::mCherryhsp70 parasites, but OVA::mCherryhsp70 parasites promoted stronger OT-I and OT-II responses than those of OVAhsp70 parasites. Despite lower OVA expression levels, OVA::Hep17hep17 parasites induced stronger T-cell responses than those of OVA::mCherryhsp70 parasites. These results indicate that unconjugated cytosolic OVA is not stably expressed in Plasmodium parasites and, importantly, that its cellular location and expression level influence both the induction and magnitude of parasite-specific T-cell responses. These parasites represent useful tools for studying the development and function of antigen-specific T-cell responses during malaria infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.

The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

Rhodamine-phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 kBT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an "order parameter," peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We describe two improved assays for in vitro and in vivo screening of inhibitors and chemicals for antimalarial activity against blood stages of the rodent malaria parasite, Plasmodium berghei. These assays are based on the determination of bioluminescence in small blood samples that is produced by reporter parasites expressing luciferase. Luciferase production increases as the parasite develops in a red blood cell and as the numbers of parasites increase during an infection. In the first assay, in vitro drug luminescence (ITDL) assay, the in vitro development of ring-stage parasites into mature schizonts in the presence and absence of candidate inhibitor(s) is quantified by measuring luciferase activity after the parasites have been allowed to mature into schizonts in culture. In the second assay, the in vivo drug luminescence (IVDL) assay, in vivo parasite growth (using a standard 4-day suppressive drug test) is quantified by measuring the luciferase activity of circulating parasites in samples of tail blood of drug-treated mice.

Genetically modified Plasmodium parasites are central gene function reagents in malaria research. The Rodent Malaria genetically modified DataBase (RMgmDB) ( www.pberghei.eu ) is a manually curated Web - based repository that contains information on genetically modified rodent malaria parasites. It provides easy and rapid access to information on the genotype and phenotype of genetically modified mutant and reporter parasites. Here, we provide guidelines for generating and describing rodent malaria parasite mutants. Standardization in describing mutant genotypes and phenotypes is important not only to enhance publication quality but also to facilitate cross-linking and mining data from multiple sources, and should permit information derived from mutant parasites to be used in integrative system biology approaches. We also provide guidelines on how to submit information to RMgmDB on non-published mutants, mutants that do not exhibit a clear phenotype, as well as negative attempts to disrupt/mutate genes. Such information helps to prevent unnecessary duplication of experiments in different laboratories, and can provide indirect evidence that these genes are essential for blood-stage development.

We describe simple and sensitive in vitro and in vivo assays to analyze Plasmodium liver stage development using transgenic P. berghei parasites (PbGFP-Luccon), which express the bioluminescent reporter protein, luciferase. In these assays, parasite development in hepatocytes is visualized and quantified by real-time bioluminescence imaging both in culture and in live mice. We also describe quantification of in vitro liver-stage development by measuring luminescence using a microplate reader. Reporter-parasite based quantification of liver-stage development is faster and correlates very well with established quantitative RT-PCR methods currently used to assess parasite development inside hepatocytes, both in live mice and in culture.

Background: Disease-causing organisms are notorious for fast rates of molecular evolution and the ability to adapt rapidly to changes in their ecology. Sex plays a key role in evolution, and recent studies, in humans and other multicellular organisms, document that genes expressed principally or exclusively in males exhibit the fastest rates of adaptive evolution. However, despite the importance of sexual reproduction for many unicellular taxa, sex-biased gene expression and its evolutionary implications have been overlooked., Methods: We analyse genomic data from multiple malaria parasite (Plasmodium) species and proteomic data sets from different parasite life cycle stages., Results: The accelerated evolution of male-biased genes has only been examined in multicellular taxa, but our analyses reveal that accelerated evolution in genes with male-specific expression is also a feature of unicellular organisms. This 'fast-male' evolution is adaptive and likely facilitated by the male-biased sex ratio of gametes in the mating pool. Furthermore, we propose that the exceptional rates of evolution we observe are driven by interactions between males and host immune responses., Conclusions: We reveal a novel form of host-parasite coevolution that enables parasites to evade host immune responses that negatively impact upon fertility. The identification of parasite genes with accelerated evolution has important implications for the identification of drug and vaccine targets. Specifically, vaccines targeting males will be more vulnerable to parasite evolution than those targeting females or both sexes.

Vaccination with live-attenuated Plasmodium sporozoites that arrest in the liver can completely protect against a malaria infection both in animal models and in humans; this has provided the conceptual basis for the most promising, but also challenging, approach to develop an efficacious malaria vaccine. Advances in genetic manipulation of Plasmodium in conjunction with improved genomic and biological information has enabled new approaches to design genetically attenuated parasites (GAPs). In this review we discuss the principles in discovery and development of GAPs in preclinical models that are important in selecting GAP parasites for first-in-human clinical studies. Finally, we highlight the challenges in manufacture, formulation and delivery of a live-attenuated whole parasite malaria vaccine, as well as the further refinements that may be implemented in the next generation GAP vaccines., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

The artificial chromosome represents a useful tool for gene transfer, both as cloning vectors and in chromosome biology research. To generate a Plasmodium artificial chromosome (PAC), we had to first functionally identify and characterize the parasite's centromere. A putative centromere (pbcen5) was cloned from chromosome 5 of the rodent parasite P. berghei based on a Plasmodium gene-synteny map. Plasmids containing pbcen5 were stably maintained in parasites during a blood-stage infection with high segregation efficiency, without drug pressure. pbcen5-containing plasmids were also stably maintained during parasite meiosis and mitosis in the mosquito. A linear PAC (L-PAC) was generated by integrating pbcen5 and telomere into a plasmid. The L-PAC segregated with a high efficiency and was stably maintained throughout the parasite's life cycle, as either one or two copies. These results suggest that L-PAC behaves like a Plasmodium chromosome, which can be exploited as an experimental research tool., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: We sought to identify the prevalence of bicuspid pulmonary valve among patients with transposition of the great arteries undergoing the arterial switch operation and evaluate functional integrity of that valve in the neoaortic position., Methods: Between October 1985 and December 2001, 391 patients had an arterial switch operation for transposition and its variants. Perioperative information and follow-up data were available for 342 patients. The serial echocardiograms of patients with bicuspid pulmonary valve were reviewed. The neoaortic valve was serially assessed, focusing on aortic insufficiency, annulus diameter, and pressure gradients., Results: Twenty-four patients (7%) had a bicuspid pulmonary valve. Age at operation was 5 days to 12 years. Two patients were lost to follow up, and 22 patients had mean follow-up of 5.3 years (range, 2 months to 13 years), of which 21 patients were alive and 1 died late. At least two postoperative echocardiogram reports were available on 19 patients. Seven patients had no neoaortic regurgitation, and 10 had trivial regurgitation. Severe aortic regurgitation developed in 1 patient with endocarditis and in another with repair of Taussig-Bing anomaly. Neoaortic valve size indexed to body surface area showed an increase in annular diameter over time proportional to somatic growth. No significant valve stenosis developed., Conclusions: Encountering a bicuspid pulmonary valve at the time of an arterial switch operation is not uncommon. The integrity of a bicuspid pulmonary valve in the neoaortic position is maintained at a mean follow-up of 5.3 years. We believe that the presence of a bicuspid pulmonary valve is not a contraindication to an arterial switch operation.

Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. The individual proteomes of male and female gametocytes were analyzed using mass spectrometry, following separation by flow sorting of transgenic parasites expressing green fluorescent protein, in a sex-specific manner. Promoter tagging in transgenic parasites confirmed the designation of stage and sex specificity of the proteins. The male proteome contained 36% (236 of 650) male-specific and the female proteome 19% (101 of 541) female-specific proteins, but they share only 69 proteins, emphasizing the diverged features of the sexes. Of all the malaria life-cycle stages analyzed, the male gametocyte has the most distinct proteome, containing many proteins involved in flagellar-based motility and rapid genome replication. By identification of gender-specific protein kinases and phosphatases and using targeted gene disruption of two kinases, new sex-specific regulatory pathways were defined.

The Molecular Approaches to Malaria 2004 meeting provided an opportunity to see the impressive progress in all research fields and in the four years since the previous Molecular Approaches to Malaria meeting, when much of the Plasmodium falciparum genome sequence was already available. Study of the part of the Plasmodium life cycle associated with transmission through the vector, which begins with the commitment of blood-stage forms to sexual development, has been especially fruitful. This success is a result of several reasons including: (i) the availability of the genome sequence; (ii) the availability of good animal models that allow parasite culture and facile in vivo studies of many of the life cycle stages involved in transmission; (iii) the availability of genetic manipulation technologies for the animal models of malaria, as well as P. falciparum; and (iv) the ability to study lethal gene knockouts at this stage of the life cycle.