Daniel J. Carucci

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.

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.

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.

Induction of CD4+ T cell-dependent CD8+ type 1 responses in humans by a malaria DNA vaccine

We assessed immunogenicity of a malaria DNA vaccine administered by needle i.m. or needleless jet injection [i.m. or i.m./intradermally (i.d.)] in 14 volunteers. Antigen-specific IFN-γ responses were detected by enzyme-linked immunospot (ELISPOT) assays in all subjects to multiple 9- to 23-aa peptides containing class I and/or class II restricted epitopes, and were dependent on both CD8+ and CD4+ T cells. Overall, frequency of response was significantly greater after i.m. jet injection. CD8+-dependent cytotoxic T lymphocytes (CTL) were detected in 8/14 volunteers. Demonstration in humans of elicitation of the class I restricted IFN-γ responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

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.

Distinct Protein Classes Including Novel Merozoite Surface Antigens in Raft-like Membranes of Plasmodium falciparum

Glycosylphosphatidylinositol (GPI)-anchored proteins coat the surface of extracellular Plasmodium falciparum merozoites, of which several are highly validated candidates for inclusion in a blood-stage malaria vaccine. Here we determined the proteome of gradient-purified detergent-resistant membranes of mature blood-stage parasites and found that these membranes are greatly enriched in GPI-anchored proteins and their putative interacting partners. Also prominent in detergent-resistant membranes are apical organelle (rhoptry), multimembrane-spanning, and proteins destined for export into the host erythrocyte cytosol. Four new GPI-anchored proteins were identified, and a number of other novel proteins that are predicted to localize to the merozoite surface and/or apical organelles were detected. Three of the putative surface proteins possessed six-cysteine (Cys6) motifs, a distinct fold found in adhesive surface proteins expressed in other life stages. All three Cys6 proteins, termed Pf12, Pf38, and Pf41, were validated as merozoite surface antigens recognized strongly by antibodies present in naturally infected individuals. In addition to the merozoite surface, Pf38 was particularly prominent in the secretory apical organelles. A different cysteine-rich putative GPI-anchored protein, Pf92, was also localized to the merozoite surface. This insight into merozoite surfaces provides new opportunities for understanding both erythrocyte invasion and anti-parasite immunity.

A DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccine Encoding Thrombospondin-Related Adhesion Protein but Not Circumsporozoite Protein Partially Protects Healthy Malaria-Naive Adults against Plasmodium falciparum Sporozoite Challenge

ABSTRACT The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-γ ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-γ ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.

Exploring the transcriptome of the malaria sporozoite stage

Most studies of gene expression in Plasmodium have been concerned with asexual and/or sexual erythrocytic stages. Identification and cloning of genes expressed in the preerythrocytic stages lag far behind. We have constructed a high quality cDNA library of the Plasmodium sporozoite stage by using the rodent malaria parasite P. yoelii, an important model for malaria vaccine development. The technical obstacles associated with limited amounts of RNA material were overcome by PCR-amplifying the transcriptome before cloning. Contamination with mosquito RNA was negligible. Generation of 1,972 expressed sequence tags (EST) resulted in a total of 1,547 unique sequences, allowing insight into sporozoite gene expression. The circumsporozoite protein (CS) and the sporozoite surface protein 2 (SSP2) are well represented in the data set. A blastx search with all tags of the nonredundant protein database gave only 161 unique significant matches (P(N) ≤ 10−4), whereas 1,386 of the unique sequences represented novel sporozoite-expressed genes. We identified ESTs for three proteins that may be involved in host cell invasion and documented their expression in sporozoites. These data should facilitate our understanding of the preerythrocytic Plasmodium life cycle stages and the development of preerythrocytic vaccines.

DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity.

Background Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. Methodology/Principal Findings The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44–817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5–102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13–408; AMA1 348, range 88–1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. Significance The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection. Trial Registration ClinicalTrials.govNCT00870987.

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

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.