Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models.

Malaria parasite evades mosquito immunity by glutaminyl cyclase-mediated posttranslational protein modification.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel.

Generation of a Genetically Modified Chimeric Parasite Expressing Circumsporozoite Protein for Malaria Vaccine Development.

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein () gene replaced with from the human parasites () and () are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing CSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric parasites expressing CSP of could be used both for clinical evaluation of vaccines targeting CSP in controlled human infections and in WSP vaccines targeting and .

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

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different ( ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard NF54 background and in a recently described Cambodian NF135.

Genome-Scale Identification of Essential Metabolic Processes for Targeting the Plasmodium Liver Stage.

Plasmodium gene functions in mosquito and liver stages remain poorly characterized due to limitations in the throughput of phenotyping at these stages. To fill this gap, we followed more than 1,300 barcoded P. berghei mutants through the life cycle.

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

Transgenic malaria parasites expressing fluorescent and bioluminescent proteins are valuable tools to interrogate malaria-parasite biology and to evaluate drugs and vaccines. Using CRISPR/Cas9 methodology a transgenic (Pf) NF54 line was generated that expresses a fusion of and genes under the control of the .3 gene promoter (line mCherry-luc@etramp10.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

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

Two members of 6-cysteine (6-cys) protein family, P48/45 and P230, are important for gamete fertility in rodent and human malaria parasites and are leading transmission blocking vaccine antigens. Rodent and human parasites encode a paralog of P230, called P230p. While P230 is expressed in male and female parasites, P230p is expressed only in male gametocytes and gametes.

Chimeric Plasmodium falciparum parasites expressing Plasmodium vivax circumsporozoite protein fail to produce salivary gland sporozoites.

Background: Rodent malaria parasites where the gene encoding circumsporozoite protein (CSP) has been replaced with csp genes from the human malaria parasites, Plasmodium falciparum or Plasmodium vivax, are used as pre-clinical tools to evaluate CSP vaccines in vivo. These chimeric rodent parasites produce sporozoites in Anopheles stephensi mosquitoes that are capable of infecting rodent and human hepatocytes. The availability of chimeric P.

OX40 Stimulation Enhances Protective Immune Responses Induced After Vaccination With Attenuated Malaria Parasites.

Protection against a malaria infection can be achieved by immunization with live-attenuated 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).

Expression of full-length Plasmodium falciparum P48/45 in P. berghei blood stages: A method to express and evaluate vaccine antigens.

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.

Comparisons between different elements of reported burden and common mental disorder in caregivers of ethnically diverse people with dementia in Trinidad.

Objective: Culture plays a significant role in determining family responsibilities and possibly influences the caregiver burden associated with providing care for a relative with dementia. This study was carried out to determine the elements of caregiver burden in Trinidadians regarding which interventions will provide the most benefit.

Methods: Seventy-five caregivers of patients diagnosed with dementia participated in this investigation.

Splenectomy to Optimize Hemoglobin S Control in Children With Sickle Cell Anemia on Chronic Transfusion Therapy for Stroke Prevention.

Chronic transfusion therapy with the goal of maintaining a hemoglobin (Hb) S <30% is the primary recommended treatment for children with sickle cell anemia and a history of overt stroke or abnormal transcranial Doppler examination. We report chronic hypersplenism as a cause of poor HbS% control in 3 children on chronic transfusion therapy for stroke prevention. Splenectomy resulted in a 39.

Protective immunity differs between routes of administration of attenuated malaria parasites independent of parasite liver load.

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.

ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model.

Background: Parasite cytoadherence within the microvasculature of tissues and organs of infected individuals is implicated in the pathogenesis of several malaria syndromes. Multiple host receptors may mediate sequestration. The identity of the host receptor(s), or the parasite ligand(s) responsible for sequestration of Plasmodium species other than Plasmodium falciparum is largely unknown.

Rational development of a protective P. vivax vaccine evaluated with transgenic rodent parasite challenge models.

Development of a protective and broadly-acting vaccine against the most widely distributed human malaria parasite, Plasmodium vivax, will be a major step towards malaria elimination. However, a P. vivax vaccine has remained elusive by the scarcity of pre-clinical models to test protective efficacy and support further clinical trials.

Signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria.

The influence of parasite genetic factors on immune responses and development of severe pathology of malaria is largely unknown. In this study, we performed genome-wide transcriptomic profiling of mouse whole blood during blood-stage infections of two strains of the rodent malaria parasite Plasmodium chabaudi that differ in virulence. We identified several transcriptomic signatures associated with the virulent infection, including signatures for platelet aggregation, stronger and prolonged anemia and lung inflammation.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P.

Correction: Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

[This corrects the article DOI: 10.1371/journal.ppat.

Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite.

Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony-is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission.

Vital and dispensable roles of Plasmodium multidrug resistance transporters during blood- and mosquito-stage development.

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P.

Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown.