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  • PublicationOpen Access
    Synthetic DNA Vaccines Adjuvanted with pIL-33 Drive Liver-Localized T Cells and Provide Protection from <i>Plasmodium</i> Challenge in a Mouse Model.
    (2020-01-10T00:00:00Z) Reeder, SM; Reuschel, EL; Bah, MA; Yun, K; Tursi, NJ; Kim, KY; Chu, J; Zaidi, FI; Yilmaz, I; Hart, RJ; Perrin, B; Xu, Z; Humeau, L; Weiner, DB; Aly, Ahmed Sayed Ibrahım; ALY, AHMED SAYED IBRAHıM
    The need for a malaria vaccine is indisputable. A single vaccine for Plasmodium pre-erythrocytic stages targeting the major sporozoite antigen circumsporozoite protein (CSP) has had partial success. Additionally, CD8+ T cells targeting liver-stage (LS) antigens induced by live attenuated sporozoite vaccines were associated with protection in human challenge experiments. To further evaluate protection mediated by LS antigens, we focused on exported pre-erythrocytic proteins (exported protein 1 (EXP1), profilin (PFN), exported protein 2 (EXP2), inhibitor of cysteine proteases (ICP), transmembrane protein 21 (TMP21), and upregulated in infective sporozoites-3 (UIS3)) expressed in all Plasmodium species and designed optimized, synthetic DNA (synDNA) immunogens. SynDNA antigen cocktails were tested with and without the molecular adjuvant plasmid IL-33. Immunized animals developed robust T cell responses including induction of antigen-specific liver-localized CD8+ T cells, which were enhanced by the co-delivery of plasmid IL-33. In total, 100% of mice in adjuvanted groups and 71%–88% in non-adjuvanted groups were protected from blood-stage disease following Plasmodium yoelii sporozoite challenge. This study supports the potential of synDNA LS antigens as vaccine components for malaria parasite infection.
  • PublicationOpen Access
    An Attenuated HSV-1-Derived Malaria Vaccine Expressing Liver-Stage Exported Proteins Induces Sterilizing Protection against Infectious Sporozoite Challenge
    (2022-02-01T00:00:00Z) Rider, Paul J. F.; KAMIL, MOHD; Yilmaz, Ilknur; Atmaca, Habibe N.; Kalkan-Yazici, Merve; Ziya Doymaz, Mehmet; Kousoulas, Konstantin G.; ALY, Ahmed Sayed Ibrahım; KAMIL, MOHD; YAZICI, MERVE; ALY, AHMED SAYED IBRAHıM
    Here, we present the construction of an attenuated herpes simplex virus type-1 (HSV-1)-vectored vaccine, expressing three liver-stage (LS) malaria parasite exported proteins (EXP1, UIS3 and TMP21) as fusion proteins with the VP26 viral capsid protein. Intramuscular and subcutaneous immunizations of mice with a pooled vaccine, composed of the three attenuated virus strains expressing each LS antigen, induced sterile protection against the intravenous challenge of Plasmodium yoelii 17X-NL salivary gland sporozoites. Our data suggest that this malaria vaccine may be effective in preventing malaria parasite infection using practical routes of immunization in humans.
  • PublicationMetadata only
    Genetic Characterization of Plasmodium Putative Pantothenate Kinase Genes Reveals Their Essential Role in Malaria Parasite Transmission to the Mosquito
    (2016-09-20T00:00:00Z) Hart, Robert J.; Cornillot, Emmanuel; Abraham, Amanah; Molina, Emily; Nation, Catherine S.; Ben Mamoun, Choukri; Aly, Ahmed Sayed Ibrahım; ALY, AHMED SAYED IBRAHıM
    The metabolic machinery for the biosynthesis of Coenzyme A (CoA) from exogenous pantothenic acid (Vitamin B-5) has long been considered as an excellent target for the development of selective antimicrobials. Earlier studies in the human malaria parasite Plasmodium falciparum have shown that pantothenate analogs interfere with pantothenate phosphorylation and block asexual blood stage development. Although two eukaryotic-type putative pantothenate kinase genes (PanK1 and PanK2) have been identified in all malaria parasite species, their role in the development of Plasmodium life cycle stages remains unknown. Here we report on the genetic characterization of PanK1 and PanK2 in P. yoelii. We show that P. yoelii parasites lacking either PanK1 or PanK2 undergo normal asexual stages development and sexual stages differentiation, however they are severely deficient in ookinete, oocyst and sporozoite formation inside the mosquito vector. Quantitative transcriptional analyses in wild-type and knockout parasites demonstrate an important role for these genes in the regulation of expression of other CoA biosynthesis genes. Together, our data provide the first genetic evidence for the importance of the early steps of pantothenate utilization in the regulation of CoA biosynthesis and malaria parasite transmission to Anopheles mosquitoes.
  • PublicationMetadata only
    SAP1 is a critical post-transcriptional regulator of infectivity in malaria parasite sporozoite stages
    (2011-02-01T00:00:00Z) Aly, Ahmed Sayed Ibrahım; Lindner, Scott E.; MacKellar, Drew C.; Peng, Xinxia; Kappe, Stefan H. I.; ALY, AHMED SAYED IBRAHıM
    Plasmodium salivary gland sporozoites upregulate expression of a unique subset of genes, collectively called the UIS (upregulated in infectious sporozoites). Many UIS were shown to be essential for early liver stage development, although little is known about their regulation. We previously identified a conserved sporozoite-specific protein, SAP1, which has an essential role in Plasmodium liver infection. Targeted deletion of SAP1 in Plasmodium yoelii caused the depletion of a number of selectively tested UIS transcripts in sporozoites, resulting in a complete early liver stage arrest. Here, we use a global gene expression survey to more comprehensively identify transcripts that are affected by SAP1 deletion. We find an effect upon both the transcript abundance of UIS genes, as well as of select genes previously not grouped as UIS. Importantly, we show that the lack of SAP1 causes the specific degradation of these transcripts. Collectively, our data suggest that SAP1 is involved in a selective post-transcriptional mechanism to regulate the abundance of transcripts critical to the infectivity of sporozoites. Although Pysap1(-) sporozoites are depleted of many of these important transcripts, they confer long-lasting sterile protection against wild-type sporozoite challenge in mice. SAP1 is therefore an appealing candidate locus for attenuation of Plasmodium falciparum.
  • PublicationMetadata only
    Single Dose Mixed Species Malaria Vaccination of Genetically Attenuated Blood Stage Malaria Parasites Induce Sterile Immunity Against Lethal Challenge by Both Species
    (2021-09-13T00:00:00Z) Deveci, Gözde; Temel, Binnur; Aly, Ahmed Sayed Ibrahım; DEVECİ, GÖZDE; TEMEL, BİNNUR; ALY, AHMED SAYED IBRAHıM
  • PublicationMetadata only
    Plasmodium yoelii Vitamin B-5 Pantothenate Transporter Candidate is Essential for Parasite Transmission to the Mosquito
    (2014-07-11T00:00:00Z) Hart, Robert J.; Lawres, Lauren; Fritzen, Emma; Ben Mamoun, Choukri; Aly, Ahmed Sayed Ibrahım; ALY, AHMED SAYED IBRAHıM
    In nearly all non-photosynthetic cells, pantothenate (vitamin B-5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.
  • PublicationOpen Access
    Automated wide-field malaria parasite infection detection using Fourier ptychography on stain-free thin-smears
    (2022-07-01T00:00:00Z) AKÇAKIR, OSMAN; Celebi, Lutfi Kadir; KAMIL, MOHD; ALY, Ahmed Sayed Ibrahım; AKÇAKIR, OSMAN; KAMIL, MOHD; ALY, AHMED SAYED IBRAHıM
    Diagnosis of malaria in endemic areas is hampered by the lack of a rapid, stain-free and sensitive method to directly identify parasites in peripheral blood. Herein, we report the use of Fourier ptychography to generate wide-field high-resolution quantitative phase images of erythrocytes infected with malaria parasites, from a whole blood sample. We are able to image thousands of erythrocytes (red blood cells) in a single field of view and make a determination of infection status of the quantitative phase image of each segmented cell based on machine learning (random forest) and deep learning (VGG16) models. Our random forest model makes use of morphology and texture based features of the quantitative phase images. In order to label the quantitative images of the cells as either infected or uninfected before training the models, we make use of a Plasmodium berghei strain expressing GFP (green fluorescent protein) in all life cycle stages. By overlaying the fluorescence image with the quantitative phase image we could identify the infected subpopulation of erythrocytes for labelling purposes. Our machine learning model (random forest) achieved 91% specificity and 72% sensitivity while our deep learning model (VGG16) achieved 98% specificity and 57% sensitivity. These results highlight the potential for quantitative phase imaging coupled with artificial intelligence to develop an easy to use platform for the rapid and sensitive diagnosis of malaria.
  • PublicationMetadata only
    Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages
    (2019-05-01T00:00:00Z) Aly, Ahmed Sayed Ibrahım; Deveci, Gozde; Yilmaz, Ilknur; Abraham, Amanah; Golshan, Aneesa; Hart, Robert J.; ALY, AHMED SAYED IBRAHıM; DEVECİ, GÖZDE
    Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P. falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites. Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.
  • PublicationMetadata only
    Plasmodium AdoMetDC/ODC bifunctional enzyme is essential for male sexual stage development and mosquito transmission
    (2016-08-15T00:00:00Z) Hart, Robert J.; Ghaffar, Atif; Abdalal, Shaymaa; Perrin, Benjamin; Aly, Ahmed Sayed Ibrahım; ALY, AHMED SAYED IBRAHıM
    Polyamines are positively-charged organic molecules that are important for cellular growth and division. Polyamines and their synthesizing enzymes are particularly abundant in rapidly proliferating eukaryotic cells such as parasitic protozoa and cancer cells. Polyamine biosynthesis inhibitors, such as Elfornithine, are now being considered for cancer prevention and have been used effectively against Trypanosoma brucei. Inhibitors of polyamine biosynthesis have caused growth arrest of Plasmodium falciparum blood stages in vitro, but in P. berghei only partial inhibition has been observed. While polyamine biosynthesis enzymes are characterized and conserved in Plasmodium spp., little is known on the biological roles of these enzymes inside malaria parasite hosts. The bifunctional polyamine biosynthesis enzyme S-adenosyl methionine decarboxylase/omithine decarboxylase (AdoMetDC/ODC) was targeted for deletion in P. yoelii. Deletion of AdoMetDC/ODC significantly reduced blood stage parasitemia but Anopheles transmission was completely blocked. We showed that male gametocytogenesis and male gamete exflagellation were abolished and consequently no ookinetes or oocyst sporozoites could be generated from adometdc/odc(-) parasites. Supplementation of putrescine and spermidine did not rescue the defective phenotypes of male gametocytes and gametes of the knockout parasites. These results highlight the crucial role of polyamine homeostasis in the development and functions of Plasmodium erythrocytic stages in the blood and in the mosquito vector and validate polyamine biosynthesis pathway enzymes as drug targeting candidates for malaria parasite transmission blocking.
  • PublicationMetadata only
    Mitochondrial Spermidine Synthase is Essential for Blood-stage growth of the Malaria Parasite
    (2022-12-01T00:00:00Z) KAMIL, MOHD; KINA, ÜMİT YAŞAR; DEVECİ, GÖZDE; Akyuz, Sevim N.; Yilmaz, Ilknur; ALY, Ahmed Sayed Ibrahım; KAMIL, MOHD; KINA, ÜMİT YAŞAR; DEVECİ, GÖZDE; ALY, AHMED SAYED IBRAHıM
    © 2022Positively-charged polyamines are essential molecules for the replication of eukaryotic cells and are particularly important for the rapid proliferation of parasitic protozoa and cancer cells. Unlike in Trypanosoma brucei, the inhibition of the synthesis of intermediate polyamine Putrescine caused only partial defect in malaria parasite blood-stage growth. In contrast, reducing the intracellular concentrations of Spermidine and Spermine by polyamine analogs caused significant defects in blood-stage growth in Plasmodium yoelii and P. falciparum. However, little is known about the synthesizing enzyme of Spermidine and Spermine in the malaria parasite. Herein, malaria parasite conserved Spermidine Synthase (SpdS) gene was targeted for deletion/complementation analyses by knockout/knock-in constructs in P. yoelii. SpdS was found to be essential for blood-stage growth. Live fluorescence imaging in blood-stages and sporozoites confirmed a specific mitochondrial localization, which is not known for any polyamine-synthesizing enzyme so far. This study identifies SpdS as an excellent drug targeting candidate against the malaria parasite, which is localized to the parasite mitochondrion.