Showing papers by "Daniel J. Carucci published in 2004"

Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle

Abstract: To investigate the role of post-transcriptional controls in the regulation of protein expression for the malaria parasite, Plasmodium falciparum, we have compared mRNA transcript and protein abundance levels for seven different stages of the parasite life cycle. A moderately high positive relationship between mRNA and protein abundance was observed for these stages; the most common discrepancy was a delay between mRNA and protein accumulation. Potentially post-transcriptionally regulated genes are identified, and families of functionally related genes were observed to share similar patterns of mRNA and protein accumulation.

A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling.

Abstract: Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.

Update on the clinical development of candidate malaria vaccines

Abstract: The recent availability of significantly increased levels of funding for unmet medical needs in the developing world, made available by newly created public-private-partnerships, has proven to be a powerful driver for stimulating clinical development of candidate vaccines for malaria. This new way forward promises to greatly increase the likelihood of bringing a safe and effective vaccine to licensure. The investigators bring together important published and unpublished information that illuminates the status of malaria vaccine development. They focus their comments on those candidate vaccines that are currently in or expected to enter clinical trials in the next 12 months.

Induction in Humans of CD8+ and CD4+ T Cell and Antibody Responses by Sequential Immunization with Malaria DNA and Recombinant Protein

Abstract: Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8 + and CD4 + T cell and Ab responses in the same individual In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8 + T cells by CTL or short-term (ex vivo) IFN-γ ELISPOT assays, and partial short-term protection P falciparum DNA vaccines elicit CD8 + T cells by these assays, but no protection We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4 + T cells, and CD8 + cytotoxic and Tc1 T cells Depending upon the immunization regime, CD4 + T cells were involved in both the induction and production phases of PfCSP-specific IFN-γ responses, whereas, CD8 + T cells were involved only in the production phase IFN-γ mRNA up-regulation was detected in both CD45RA − (CD45RO + ) and CD45RA + CD4 + and CD8 + T cell populations after stimulation with PfCSP peptides This finding suggests CD45RA + cells function as effector T cells The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines

Proteome analysis of rhoptry-enriched fractions isolated from Plasmodium merozoites.

Abstract: The rhoptries of Plasmodium species participate in merozoite invasion and modification of the host erythrocyte. However, only a few rhoptry proteins have been identified using conventional gene ide...

Reduced immunogenicity of DNA vaccine plasmids in mixtures.

Abstract: We measured the ability of nine DNA vaccine plasmids encoding candidate malaria vaccine antigens to induce antibodies and interferon-γ responses when delivered alone or in a mixture containing all nine plasmids. We further examined the possible immunosuppressive effect of individual plasmids, by assessing a series of mixtures in which each of the nine vaccine plasmids was replaced with a control plasmid. Given alone, each of the vaccine plasmids induced significant antibody titers and, in the four cases for which appropriate assays were available, IFN-γ responses. Significant suppression or complete abrogation of responses were seen when the plasmids were pooled in a nine-plasmid cocktail and injected in a single site. Removal of single genes from the mixture frequently reduced the observed suppression. Boosting with recombinant poxvirus increased the antibody response in animals primed with either a single gene or the mixture, but, even after boosting, responses were higher in animals primed with single plasmids than in those primed with the nine-plasmid mixture. Boosting did not overcome the suppressive effect of mixing for IFN-γ responses. Interactions between components in a multiplasmid DNA vaccine may limit the ability to use plasmid pools alone to induce responses against multiple targets simultaneously.

High-throughput generation of P. falciparum functional molecules by recombinational cloning.

Abstract: Large-scale functional genomics studies for malaria vaccine and drug development will depend on the generation of molecular tools to study protein expression. We examined the feasibility of a high-throughput cloning approach using the Gateway system to create a large set of expression clones encoding Plasmodium falciparum single-exon genes. Master clones and their ORFs were transferred en masse to multiple expression vectors. Target genes (n = 303) were selected using specific sets of criteria, including stage expression and secondary structure. Upon screening four colonies per capture reaction, we achieved 84% cloning efficiency. The genes were subcloned in parallel into three expression vectors: a DNA vaccine vector and two protein expression vectors. These transfers yielded a 100% success rate without any observed recombination based on single colony screening. The functional expression of 95 genes was evaluated in mice with DNA vaccine constructs to generate antibody against various stages of the parasite. From these, 19 induced antibody titers against the erythrocytic stages and three against sporozoite stages. We have overcome the potential limitation of producing large P. falciparum clone sets in multiple expression vectors. This approach represents a powerful technique for the production of molecular reagents for genome-wide functional analysis of the P. falciparum genome and will provide for a resource for the malaria resource community distributed through public repositories.

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei.

Abstract: Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.

Know thine enemy.

Abstract: The malaria and mosquito genomes will allow us to find new drug and vaccine targets, says Daniel Carucci.

Effect on antibody and T-cell responses of mixing five GMP-produced DNA plasmids and administration with plasmid expressing GM-CSF.

Abstract: One potential benefit of DNA vaccines is the capacity to elicit antibody and T-cell responses against multiple antigens at the same time by mixing plasmids expressing different proteins. A possible negative effect of such mixing is interference among plasmids regarding immunogenicity. In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone. The mixture induced higher levels of antibodies against whole parasites than did the individual plasmids, but was associated with a decrease in antibodies to individual P. falciparum proteins. T-cell responses were in general decreased by administration of the mixture. Immune responses to individual plasmids and mixtures were generally higher in inbred mice than in outbreds. In inbred BALB/c and C57BL/6 mice, coadministration of a plasmid expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), increased antibody and T-cell responses, but in outbred CD-1 mice, coadministration of mGM-CSF was associated with a decrease in antibody responses. Such variability in data from studies in different strains of mice underscores the importance of genetic background on immune response and carefully considering the goals of any preclinical studies of vaccine mixtures planned for human trials.

Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

Abstract: We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

Molecular approaches to malaria

Abstract: Malaria is a serious health problem in developing countries. With the complete sequencing of the genomes of the parasite and of the mosquito vector, malaria research has entered the post-genome era. In this report we summarize the results and new research avenues presented at a recent meeting held with the aim of developing interdisciplinary approaches to combat this disease.

A Small Peptide (CEL-1000) Derived from the β-Chain of the Human Major Histocompatibility Complex Class II Molecule Induces Complete Protection against Malaria in an Antigen-Independent Manner

Abstract: CEL-1000 (DGQEEKAGVVSTGLIGGG) is a novel potential preventative and therapeutic agent. We report that CEL-1000 confers a high degree of protection against Plasmodium sporozoite challenge in a murine model of malaria, as shown by the total absence of blood stage infection following challenge with 100 sporozoites (100% protection) and by a substantial reduction (400-fold) of liver stage parasite RNA following challenge with 50,000 sporozoites. CEL-1000 protection was demonstrated in A/J (H-2 a ) and C3H/HeJ (H-2 k ) mice but not in BALB/c (H-2 d ) or CAF1 (A/J BALB/c F1 hybrid) mice. In CEL-1000-treated and protected mice, high levels of gamma interferon (IFN-) in serum and elevated frequencies of hepatic and splenic CD4 IFNpositive T cells were detected 24 h after administration of an additional dose of CEL-1000. Treatment of A/J mice that received CEL-1000 with antibodies against IFN- just prior to challenge abolished the protection, and a similar treatment with antibodies against CD4 T cells partially reduced the level of protection, while treatment with control antibodies or antibodies specific for interleukin-12 (IL-12), CD8 T cells, or NK cells had no effect. Our data establish that the protection induced by CEL-1000 is dependent on IFN- and is partially dependent on CD4 T cells but is independent of CD8 T cells, NK cells, and IL-12 at the effector phase and does not induce a detectable antibody response. Malaria remains a major cause of mortality and morbidity in tropical and subtropical areas of the world, with approximately 300 million people considered at risk of infection. Although several clinical malaria vaccines have been developed and are being evaluated for their protective efficacies (for a review, see reference 19), prophylaxis and treatment with antimalarial drugs remain the only options for effective malaria control. In recent years, drug-resistant parasites have emerged and are now widespread, presenting serious problems for malaria control. Therefore, new and effective antimalarial interventions, both drugs and vaccines, are needed. It was previously dem

Malaria Parasites: Genomes and Molecular Biology

Abstract: The completion of the Plasmodium falciparum genome sequence in late 2002 hearlded a new era in malaria research. The search began in earnest for new drugs and vaccines to combat malaria, a disease which afflicts up to 500 million people worldwide and is responsible for the deaths of more than one million people each year. The new genomic data is aiding a greater understanding of the living parasite and its interaction with the insect vector and human host.