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Daniel J Carucci

Publications and source records attributed to Daniel J Carucci.

At least 37 records · Page 2Linked to original sources
PubMed2003

Discovery of gene function by expression profiling of the malaria parasite life cycle.

The completion of the genome sequence for Plasmodium falciparum, the species responsible for most malaria human deaths, has the potential to reveal hundreds of new drug targets and proteins involved in pathogenesis. However, only approximately 35% of the genes code for proteins with an identifiable function. The absence of routine genetic tools for studying Plasmodium parasites suggests that this number is unlikely to change quickly if conventional serial methods are used to characterize encoded proteins. Here, we use a high-density oligonucleotide array to generate expression profiles of human and mosquito stages of the malaria parasite's life cycle. Genes with highly correlated levels and temporal patterns of expression were often involved in similar functions or cellular processes.

Animals↗
PubMed2003

Identification of Plasmodium falciparum antigens by antigenic analysis of genomic and proteomic data.

The recent explosion in genomic sequencing has made available a wealth of data that can now be analyzed to identify protein antigens, potential targets for vaccine development. Here we present, in the context of Plasmodium falciparum, a strategy that rapidly identifies target antigens from large and complex genomes. Sixteen antigenic proteins recognized by volunteers immunized with radiation-attenuated P. falciparum sporozoites, but not by mock immunized controls, were identified. Several of these were more antigenic than previously identified and well characterized P. falciparum-derived protein antigens. The data suggest that immune responses to Plasmodium are dispersed on a relatively large number of parasite antigens. These studies have implications for our understanding of immunodominance and breadth of responses to complex pathogens.

Adult↗
PubMed2003

A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encoding Plasmodium falciparum sexual-stage antigen, Pfs230.

Mammalian expression vectors encoding region C of malaria transmission-blocking vaccine candidate Pfs230 (aa 443-1132) with and without a 3' glycosylphosphatidylinositol (GPI) anchor signal sequence were tested for their immunogenicity in mice. The plasmid containing the GPI anchor signal sequence consistently induced higher titers of anti-Pfs230 antibodies using three delivery systems: intramuscular (i.m.), intradermal (i.d.), and gene gun (g.g.). In contrast, the isotype profile elicited varied depending on the delivery system and was not effected by the presence of the GPI anchor sequence. Both gene gun and intradermal administration induced primarily an IgG1 response, while intramuscular injection induced both IgG1 and IgG2a antibodies. Regardless of the mode of delivery, all the plasmids encoding Pfs230 region C primed for a mixed IgG1/IgG2a response to an intraperitoneal (i.p.) injection of E. coli-produced recombinant Pfs230 region C. None of these vaccination strategies were more effective than r230/MBP.C alone in generating malaria transmission-blocking immunity.

Animals↗
PubMed2003

PfSPATR, a Plasmodium falciparum protein containing an altered thrombospondin type I repeat domain is expressed at several stages of the parasite life cycle and is the target of inhibitory antibodies.

The annotated sequence of chromosome 2 of Plasmodium falciparum was examined for genes encoding proteins that may be of interest for vaccine development. We describe here the characterization of a protein with an altered thrombospondin Type I repeat domain (PfSPATR) that is expressed in the sporozoite, asexual, and sexual erythrocytic stages of the parasite life cycle. Immunoelectron microscopy indicated that this protein was expressed on the surface of the sporozoites and around the rhoptries in the asexual erythrocytic stage. An Escherichia coli-produced recombinant form of the protein bound to HepG2 cells in a dose-dependent manner and antibodies raised against this protein blocked the invasion of sporozoites into a transformed hepatoma cell line. Sera from Ghanaian adults and from a volunteer who had been immunized with radiation-attenuated P. falciparum sporozoites specifically recognized the expression of this protein on transfected COS-7 cells. These data support the evaluation of this protein as a vaccine candidate.

Amino Acid Sequence↗
PubMed2002

Exploring the proteome of Plasmodium.

With the entire genomic sequence of several species of Plasmodium soon to be available, researchers are now focusing on methods to study gene and protein expression at the whole organism level. Traditional methods of characterising and identifying large numbers of proteins from a complex protein mixture have relied predominantly on two-dimensional gel electrophoresis combined with N-terminal sequencing or mass spectrometry of individually prepared proteins. New proteomics methods are now available that are based on resolving small peptides derived from complex protein mixtures by high-resolution liquid chromatography and directly identifying them by tandem mass spectrometry (LC/LC/MS/MS) and sophisticated computer search algorithms against whole genome sequence databases. These newer proteomic methods have the potential to accelerate the reproducible identification of large numbers of proteins from various life cycle stages of Plasmodium and may help to better understand parasite biology and lead to the identification of new targets of vaccines and drugs.

Animals↗
PubMed2002

Sequence of Plasmodium falciparum chromosomes 2, 10, 11 and 14.

The mosquito-borne malaria parasite Plasmodium falciparum kills an estimated 0.7-2.7 million people every year, primarily children in sub-Saharan Africa. Without effective interventions, a variety of factors-including the spread of parasites resistant to antimalarial drugs and the increasing insecticide resistance of mosquitoes-may cause the number of malaria cases to double over the next two decades. To stimulate basic research and facilitate the development of new drugs and vaccines, the genome of Plasmodium falciparum clone 3D7 has been sequenced using a chromosome-by-chromosome shotgun strategy. We report here the nucleotide sequences of chromosomes 10, 11 and 14, and a re-analysis of the chromosome 2 sequence. These chromosomes represent about 35% of the 23-megabase P. falciparum genome.

Animals↗
PubMed2002

Genome sequence of the human malaria parasite Plasmodium falciparum.

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

Animals↗
PubMed2002

Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii.

Species of malaria parasite that infect rodents have long been used as models for malaria disease research. Here we report the whole-genome shotgun sequence of one species, Plasmodium yoelii yoelii, and comparative studies with the genome of the human malaria parasite Plasmodium falciparum clone 3D7. A synteny map of 2,212 P. y. yoelii contiguous DNA sequences (contigs) aligned to 14 P. falciparum chromosomes reveals marked conservation of gene synteny within the body of each chromosome. Of about 5,300 P. falciparum genes, more than 3,300 P. y. yoelii orthologues of predominantly metabolic function were identified. Over 800 copies of a variant antigen gene located in subtelomeric regions were found. This is the first genome sequence of a model eukaryotic parasite, and it provides insight into the use of such systems in the modelling of Plasmodium biology and disease.

Animals↗
PubMed2002

A proteomic view of the Plasmodium falciparum life cycle.

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.

Animals↗
PubMed2002

Transcripts of developmentally regulated Plasmodium falciparum genes quantified by real-time RT-PCR.

Plasmodium falciparum intraerythrocytic development is a complex process. Development proceeds rapidly from the trophozoite phase of nutrient acquisition and growth through to the synthetic and reproductive schizont phase, which ends with production of new invasive merozoites. During this process, the malaria parasite must express a series of different gene products, depending on its metabolic and synthetic needs. We are particularly interested in the development of the merozoite's organelles in the apical complex, which form during the later schizont stages. We have used quantitative real-time RT-PCR fluorogenic 5' nuclease assays (TaqMan) for the first time on malaria parasites for analysis of erythrocytic stage-specific gene expression. We analyzed transcripts of the P.falciparum eba-175 and other erythrocyte binding-like (ebl) family genes in temperature-synchronized parasites and found ebl genes have tightly controlled, stage-specific transcription. As expected, eba-175 transcripts were abundant only at the end of schizont development in a pattern most common among ebl, including baebl, pebl and jesebl. The maebl transcript pattern was unique, peaking at mid-late trophozoite stage, but absent in late-stage schizonts. ebl-1 demonstrated another pattern of expression, which peaked during mid-schizont stage and then significantly diminished in late-stage schizonts. Our analysis demonstrates that using real-time RT-PCR fluorogenic 5' nuclease assays is a sensitive, quantitative method for analysis of Plasmodium transcripts.

Animals↗
PubMed2002

Technologies for the study of gene and protein expression in Plasmodium.

With the imminent completion of the genome sequences of several species of Plasmodium, attention is now turning to the exploitation of these genomic sequence data for vaccine, drug and diagnostic development. Several technologies have been developed over the past decade to assist in the determination of gene and protein expression on a global scale. Of these, DNA microarrays, novel high-throughput proteomic technologies and recombinational cloning technologies are lowering the barrier to functional genomic studies in Plasmodium. Of equal importance is the capacity to manipulate, store, retrieve and analyse the tremendous quantity of data generated from these genomic studies. This paper will address the use of these technologies as well as some of the computational tools that will be ultimately required to adequately study gene and protein expression in Plasmodium.

Animals↗
PubMed2002

A multilateral effort to develop DNA vaccines against falciparum malaria.

Scientists from several organizations worldwide are working together to develop a multistage, multigene DNA-based vaccine against Plasmodium falciparum malaria. This collaborative vaccine development effort is named Multi-Stage DNA-based Malaria Vaccine Operation. An advisory board of international experts in vaccinology, malariology and field trials provides the scientific oversight to support the operation. This article discusses the rationale for the approach, underlying concepts and the pre-clinical development process, and provides a brief outline of the plans for the clinical testing of a multistage, multiantigen malaria vaccine based on DNA plasmid immunization technology.

Animals↗
PubMed2002

Persistence of protective immunity to malaria induced by DNA priming and poxvirus boosting: characterization of effector and memory CD8(+)-T-cell populations.

The persistence of immunity to malaria induced in mice by a heterologous DNA priming and poxvirus boosting regimen was characterized. Mice were immunized by priming with DNA vaccine plasmids encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) and murine granulocyte-macrophage colony-stimulating factor and boosting with recombinant vaccinia encoding PyCSP. BALB/c mice immunized with either high-dose (100 microg of p PyCSP plus 30 microg of pGM-CSF) or low-dose (1 microg of p PyCSP plus 1 microg of pGM-CSF DNA) priming were protected against challenge with 50 P. yoelii sporozoites. Protection 2 weeks after immunization was 70 to 100%, persisted at this level for at least 20 weeks, and declined to 30 to 40% by 28 weeks. Eight of eight mice protected at 20 weeks were still protected when rechallenged at 40 weeks. The antigen (Ag)-specific effector CD8(+)-T-cell population present 2 weeks after boosting had ex vivo Ag-specific cytolytic activity, expressed both gamma interferon (IFN-gamma) and tumor necrosis factor alpha, and constituted 12 to 20% of splenic CD8(+) T cells. In contrast, the memory CD8(+)-Ag-specific-cell population at 28 weeks lacked cytolytic activity and constituted only 6% of splenic CD8(+) T cells, but at the single-cell level it produced significantly higher levels of IFN-gamma than the effectors. High levels of Ag- or parasite-specific antibodies present 2 weeks after boosting had declined three- to sevenfold by 28 weeks. Low-dose priming was similarly immunogenic and as protective as high-dose priming against a 50-, but not a 250-, sporozoite challenge. These results demonstrate that a heterologous priming and boosting vaccination can provide lasting protection against malaria in this model system.

Amino Acid Sequence↗
PubMed2002

Optimal induction of antigen-specific CD8+ T cell responses requires bystander cell participation.

Efficient activation of specific immune responses requires a concerted interaction between T cells and antigen-presenting cells. A requirement for bystander participation of CD4+ T cells for expansion and maintenance of memory CD8+ T cells has been noted in several models, but a role with regard to effector CD8+ T responses has not been well-defined. In this report, the requirement of bystander participation for optimal induction of antigen-specific CD8+ T cell effector function was determined by directly quantitating antigen-specific interferon-gamma (IFN-gamma) CD8+ T cell responses by enzyme-linked immunospot assays, and by indirectly evaluating induction of the chemokine monokine induced by IFN-gamma as a marker for IFN-gamma-mediated effector function. Our results demonstrate that bystander cell participation, mediated by CD4+ T cell and natural killer (NK) cells, is required for optimal induction of antigen-specific CD8+ T cell effector responses. Our data further establish a novel role for NK cells in the activation of antigen-specific immune responses.

Adult↗
PubMedDate not supplied

Proteomics in malaria.

The recent completion of human, Anopheles gambiae, and Plasmodium falciparum genomes relevant to the study of human malaria allows the application of modern proteomic technologies to complement previously implemented conventional approaches. Proteomic analysis has been employed to elucidate global protein expression profiles, subcellular localization of gene products, and host-pathogen interactions that are central to disease pathogenesis and treatment. The high-throughput nature of these techniques is in accord with the pace of drug and vaccine development that have the potential to directly reduce the morbidity and mortality of disease.

Animals↗