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Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission for host to mosquito vector. Of all the malaria life cycles stages analyzed the male gametocyte has the most distinct proteome, containing many proteins involved in flagellar-based motility and rapid genome replication.

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Abstract: Invasive forms of apicomplexan parasites contain secretory organelles (which may include micronemes, rhoptries or dense granules), the contents of which mediate invasion of host cells. Only few rhoptry proteins have been identified in Plasmodium and then only in merozoites and none in the sporozoite or ookinete. Epitope-tagged proteins (with either green fluorescent protein or C-MYC) were used to analyse the expression and cellular localisation of a known Plasmodium rhoptry protein (RAP2/3) and putative homologues of two Toxoplasma rhoptry proteins (rhoptry neck protein 2, RON2 and putative rhoptry protein 2, PRP2) at different stages across the life cycle. This analysis showed correct targeting to the merozoite rhoptries of GFP-tagged RAP2/3 and, for the first time, a distinct apical fluorescence pattern in sporozoites indicating a rhoptry location. In addition, tagged PBRON2 and PBPRP2 also show a merozoite rhoptry localisation similar to that of RAP2/3. RON2 is expressed in sporozoites and has the same timing of expression and location as RAP2/3. While PRP2 is also expressed in sporozoites, both its pattern of expression and location differ from RON2 and RAP2/3. None of the tagged proteins were detected in ookinetes, which is in agreement with the proposed lack of rhoptries in this third invasive form of Plasmodium. The analysis of tagged rhoptry proteins reveals new insights into the role of these proteins in host-cell invasion in different malarial ‘zoites’ and will facilitate more detailed studies into the role of rhoptries in establishing an infection of not only red blood cell but also the hepatocytes. [Copyright &y& Elsevier]

Highlights • Full-length Pfs48/45 protein is efficiently expressed in blood stages of P. berghei. • P. berghei blood-stage expressed Pfs48/45 retain conformational epitopes. • Strong transmission blocking immunity is induced by P. berghei expressed Pfs48/45. Abstract The transmission-blocking vaccine candidate Pfs48/45 from the human malaria parasite Plasmodium falciparum is known to be difficult to express in heterologous systems, either as full-length protein or as correctly folded protein fragments that retain conformational epitopes. In this study we express full-length Pfs48/45 in the rodent parasite P. berghei. Pfs48/45 is expressed as a transgene under control of the strong P. berghei schizont-specific msp1 gene promoter (Pfs48/45@PbMSP1). Pfs48/45@PbMSP1 schizont-infected red blood cells produced full-length Pfs48/45 and the structural integrity of Pfs48/45 was confirmed using a panel of conformation-specific monoclonal antibodies that bind to different Pfs48/45 epitopes. Sera from mice immunized with transgenic Pfs48/45@PbMSP1 schizonts showed strong transmission-reducing activity in mosquitoes infected with P. falciparum using standard membrane feeding. These results demonstrate that transgenic rodent malaria parasites expressing human malaria antigens may be used as means to evaluate immunogenicity and functionality of difficult to express malaria vaccine candidate antigens. [ABSTRACT FROM AUTHOR]

The parasitic disease malaria threatens more than 3 billion people worldwide, resulting in more than 200 million clinical cases and almost 600,000 deaths annually. Vaccines remain crucial for prevention and ultimately eradication of infectious diseases and, for malaria, whole sporozoite based immunization has been shown to be the most effective in experimental settings. In addition to immunization with radiation-attenuated sporozoites, chemoprophylaxis and sporozoites (CPS) is a highly efficient strategy to induce sterile protection in humans. Genetically attenuated parasites (GAP) have demonstrated significant protection in rodent studies, and are now being advanced into clinical testing. This review describes the existing pre-clinical and clinical data on CPS and GAP, discusses recent developments and examines how to transform these immunization approaches into vaccine candidates for clinical development. [ABSTRACT FROM AUTHOR]

Abstract: Splenomegaly, albeit variably, is a hallmark of malaria; yet, the role of the spleen in Plasmodium infections remains vastly unknown. The implementation of imaging to study the spleen is rapidly advancing our knowledge of this so-called “blackbox” of the abdominal cavity. Not only has ex vivo imaging revealed the complex functional compartmentalization of the organ and immune effector cells, but it has also allowed the observation of major structural remodeling during infections. In vivo imaging, on the other hand, has allowed quantitative measurements of the dynamic passage of the parasite at spatial and temporal resolution. Here, we review imaging techniques used for studying the malarious spleen, from optical microscopy to in vivo imaging, and discuss the bright perspectives of evolving technologies in our present understanding of the role of this organ in infections caused by Plasmodium. [Copyright &y& Elsevier]

Highlights: [•] We use 2 transgenic rodent malaria models to visualize parasite liver load in vivo. [•] We show that multiple variables contribute to the parasite liver load. [•] IM injections result in a higher liver load compared to ID or SC injections [•] Our findings provide a rationale for the design of clinical trials. [ABSTRACT FROM AUTHOR]

Abstract: Background: Stress-only myocardial perfusion imaging (MPI) saves time by eliminating rest imaging, which is important for emergency department (ED) throughput but has not been studied in an ED population. Study Objective: To determine the prognosis of a normal stress-only MPI study compared to a normal rest-stress MPI and establish its effectiveness in an ED setting. Methods: All patients evaluated in the ED over 6.5 years who underwent a stress-only technetium-99m gated MPI were compared to those who had a rest-stress study. All-cause mortality was determined using the Social Security Death Index. Survival was analyzed in patients with normal and abnormal MPI results. Results: A total of 4145 studies (2340 stress-only, 1805 rest-stress) were performed. Patients’ average age was 57.9 years, 38.5% were male, and most had an intermediate or low pretest risk of coronary artery disease (87.7%). Average follow-up was 35.9 ± 20.9 months. In patients with normal perfusion, at 1 year of follow-up there were 11 deaths in the stress-only group (0.5% 1-year mortality), and 13 deaths in the rest-stress cohort (1.1% 1-year mortality). At the end of follow-up, the stress-only group had a lower all-cause mortality (p < 0.0001) and similar risk adjusted all-cause mortality (p = 0.10) than the rest-stress cohort. Patients with abnormal perfusion demonstrated the expected differential prognosis based on total perfusion deficits in both groups. Conclusions: A normal stress-only MPI study has a benign 1-year prognosis similar to a rest-stress study when performed in the ED. The ability to triage patients more rapidly and reduce radiation exposure represents an attractive alternative for low-risk patients. [Copyright &y& Elsevier]

Abstract: The critical first step in the clinical development of a malaria vaccine, based on live-attenuated Plasmodium falciparum sporozoites, is the guarantee of complete arrest in the liver. We report on an approach for assessing adequacy of attenuation of genetically attenuated sporozoites in vivo using the Plasmodium berghei model of malaria and P. falciparum sporozoites cultured in primary human hepatocytes. We show that two genetically attenuated sporozoite vaccine candidates, Δp52+p36 and Δfabb/f, are not adequately attenuated. Sporozoites infection of mice with both P. berghei candidates can result in blood infections. We also provide evidence that P. falciparum sporozoites of the leading vaccine candidate that is similarly attenuated through the deletion of the genes encoding the proteins P52 and P36, can develop into replicating liver stages. Therefore, we propose a minimal set of screening criteria to assess adequacy of sporozoite attenuation necessary before advancing into further clinical development and studies in humans. [Copyright &y& Elsevier]

The RMgm database, www.pberghei.eu, is a web-based, manually curated, repository containing information on genetically modified rodent-malaria parasites. It provides easy and rapid access to information on the genotype and phenotype of mutant and reporter parasites. The database also contains information on unpublished mutants without a clear phenotype and negative trials to disrupt genes. Information can be searched using pre-defined key features, such as phenotype, life-cycle stage, gene model, gene-tags and mutations. The information relating to the mutants is reciprocally linked to PlasmoDB and GeneDB. Access to mutant-parasite information, and gene function/ontology inferred from mutant phenotypes provides a timely resource aimed at enhancing research into Plasmodium gene function and (systems) biology. [ABSTRACT FROM AUTHOR]

Abstract: Biological cells are complex and highly dynamic: many macromolecules are organized in loose assemblies, clusters or highly structured complexes, others exist most of the time as freely diffusing monomers. They move between regions and compartments through diffusion and enzyme-mediated transport, within a heavily crowded cytoplasm. To make sense of this complexity, computational models, and, in turn, quantitative in vivo data are needed. An array of fluorescent microscopy methods is available, but due to the inherent noise and complexity inside the cell, they are often hard to interpret. Using the example of fluorescence recovery after photobleaching (FRAP) and the bacterial chemotaxis system, we are here introducing detailed spatial simulations as a new approach in analysing such data. [Copyright &y& Elsevier]