The Anopheles gambiae adult midgut peritrophic matrix proteome.

Malaria is a devastating disease. For transmission to occur, Plasmodium, the causative agent of malaria, must complete a complex developmental cycle in its mosquito vector. Thus, the mosquito is a potential target for disease control. Plasmodium ookinetes, which develop within the mosquito midgut, must first cross the midgut's peritrophic matrix (PM), a thick extracellular sheath that completely surrounds the blood meal. The PM poses a partial, natural barrier against parasite invasion of the midgut and it is speculated that modifications to the PM may lead to a complete barrier to infection. However, such strategies require thorough characterization of the structure of the PM. Here, we describe for the first time, the complete PM proteome of the main malaria vector, Anopheles gambiae. Altogether, 209 proteins were identified by mass spectrometry. Among them were nine new chitin-binding peritrophic matrix proteins, expanding the list from three to twelve peritrophins. Lastly, we provide a model for the putative interactions among the proteins identified in this study.

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.

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-gamma) 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-gamma 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-gamma 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.

Functional proteome and expression analysis of sporozoites and hepatic stages of malaria development.

An evolution in modern malaria research occurred with the completion of the Plasmodium falciparum genome project and the onset and application of novel post-genomic technologies. Corresponding with these technological achievements are improvements in accessing and purifying parasite material from 'hard-to-reach' stages of malaria development. Characterization of gene and protein expression in the infectious sporozoite and subsequent liver-stage parasite development is critical to identify novel pre-erythrocytic drug and vaccine targets as well as to understand the basic biology of this deadly parasite. Both transcriptional and proteomic analyses on these stages and the remaining stages of development will assist in the 'credentialing process' of the complete malaria genome.

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

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.

Proteomic approaches to studying drug targets and resistance in Plasmodium.

Ever increasing drug resistance by Plasmodium falciparum, the most virulent of human malaria parasites, is creating new challenges in malaria chemotherapy. The entire genome sequences of P. falciparum and the rodent malaria parasite, P. yoelii yoelii are now available. Extensive genome sequence data from other Plasmodium species including another important human malaria parasite, P. vivax are also available. Powerful research techniques coupled to genomic resources are needed to help identify new drug and vaccine targets against malaria. Applied to Plasmodium, proteomics combines high-resolution protein or peptide separation with mass spectrometry and computer software to rapidly identify large numbers of proteins expressed from various stages of parasite development. Proteomic methods can be applied to study sub-cellular localization, cell function, organelle composition, changes in protein expression patterns in response to drug exposure, drug-protein binding and validation of data from genomic annotation and transcript expression studies. Recent high-throughput proteomic approaches have provided a wealth of protein expression data on P. falciparum, while smaller-scale studies examining specific drug-related hypotheses are also appearing. Of particular interest is the study of mechanisms of action and resistance of drugs such as the quinolines, whose targets currently may not be predictable from genomic data. Coupling the Plasmodium sequence data with bioinformatics, proteomics and RNA transcript expression profiling opens unprecedented opportunities for exploring new malaria control strategies. This review will focus on pharmacological research in malaria and other intracellular parasites using proteomic techniques, emphasizing resources and strategies available for Plasmodium.

Reduced immunogenicity of DNA vaccine plasmids in mixtures.

We measured the ability of nine DNA vaccine plasmids encoding candidate malaria vaccine antigens to induce antibodies and interferon-gamma 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-gamma 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-gamma 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.

Utilization of genomic sequence information to develop malaria vaccines.

Recent advances in the fields of genomics, proteomics and molecular immunology offer tremendous opportunities for the development of novel interventions against public health threats, including malaria. However, there is currently no algorithm that can effectively identify the targets of protective T cell or antibody responses from genomic data. Furthermore, the identification of antigens that will stimulate the most effective immunity against the target pathogen is problematic, particularly if the genome is large. Malaria is an attractive model for the development and validation of approaches to translate genomic information to vaccine development because of the critical need for effective anti-malarial interventions and because the Plasmodium parasite is a complex multistage pathogen targeted by multiple immune responses. Sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites, and anti-disease immunity can be induced in residents in malaria-endemic areas. However, the 23 Mb Plasmodium falciparum genome encodes more than 5,300 proteins, each of which is a potential target of protective immune responses. The current generation of subunit vaccines is based on a single or few antigens and therefore might elicit too narrow a breadth of response. We are working towards the development of a new generation vaccine based on the presumption that duplicating the protection induced by the whole organism may require a vaccine nearly as complex as the organism itself. Here, we present our strategy to exploit the genomic sequence of P. falciparum for malaria vaccine development.

The infectivity of Plasmodium yoelii in different strains of mice.

We evaluated the effect of using Medium 199 alone and Medium 199 supplemented with 5% normal mouse serum, 5% fetal calf serum, 5% bovine serum albumin or 5% Albumax on Plasmodium yoelii sporozoite yield from infected mosquitoes and infectivity in BALB/c mice. The sporozoites yield, as well as their infectivity, was statistically lower (P = 0.0031) when unsupplemented Medium 199 was used to separate sporozoites from infected mosquitoes. Although Medium 199 supplemented with Albumax led to lower sporozoite yield (P < 0.0009), infectivity of the sporozoites was similar to those obtained with the other medium supplements. Because normal mouse serum supports good sporozoite infections and is also the supplement that can be used repeatedly in mice during multiple sporozoite injections without inducing anaphylaxis, we selected it to evaluate the infectivity of P. yoelii sporozoites in different strains of mice. After injecting mice with serial dilutions of sporozoites and detecting patent infections, we determined that the infective dose 50 (ID50) for BALB/c, C57Bl/6, A/J, and B10BR mice ranged between 4.9 and 10.6 sporozoites. The ID50 obtained for CD-1 mice (147 sporozoites) was significantly higher.

Determining liver stage parasite burden by real time quantitative PCR as a method for evaluating pre-erythrocytic malaria vaccine efficacy.

The detection and quantitation of blood stage parasitaemia is typically used as a surrogate endpoint for estimating the efficacy of vaccines targeted against the hepatic stage, as well as the erythrocytic stage, of the parasite. However, this does not provide an adequate means of evaluating the efficacy of vaccines, which may be only partially effective at the liver-stage. This is a particular concern for effective evaluation of immune enhancement strategies for candidate pre-erythrocytic stage vaccines. Here, we have developed and validated a method for detecting and quantitating liver stage parasites, using the TaqMan fluorescent real-time quantitative PCR system (PE Applied Biosystems). This method uses TaqMan primers designed to the Plasmodium yoelii 18S rRNA gene and rodent GAPDH to amplify products from infected mouse liver cDNA. The technique is highly reproducible as demonstrated with plasmid controls and capable of efficiently quantitating liver-stage parasite burden following a range of sporozoite challenge doses in strains of mice, which differ in their susceptibility to sporozoite infection. We have further demonstrated the capacity of this technique to evaluate the efficacy of a range of pre-erythrocytic stage vaccines. Our data establish this quantitative real-time PCR assay to be a fast and reproducible way of accurately assessing liver stage parasite burden and vaccine efficacy in rodent malaria models.

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-gamma 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-gamma responses we believe necessary for protection against the liver stage of malaria parasites brings us closer to an effective malaria vaccine.

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) < or = 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.

Immunogenicity and protective efficacy of a Plasmodium yoelii Hsp60 DNA vaccine in BALB/c mice.

The gene encoding the 60-kDa heat shock protein of Plasmodium yoelii (PyHsp60) was cloned into the VR1012 and VR1020 mammalian expression vectors. Groups of 10 BALB/c mice were immunized intramuscularly at 0, 3, and 9 weeks with 100 microg of PyHsp60 DNA vaccine alone or in combination with 30 microg of pmurGMCSF. Sera from immunized mice but not from vector control groups recognized P. yoelii sporozoites, liver stages, and infected erythrocytes in an indirect fluorescent antibody test. Two weeks after the last immunization, mice were challenged with 50 P. yoelii sporozoites. In one experiment the vaccine pPyHsp60-VR1012 used in combination with pmurGMCSF gave 40% protection (Fisher's exact test; P = 0.03, vaccinated versus control groups). In a second experiment this vaccine did not protect any of the immunized mice but induced a delay in the onset of parasitemia. In neither experiment was there any evidence of a protective effect against the asexual erythrocytic stage of the life cycle. In a third experiment mice were primed with PyHsp60 DNA, were boosted 2 weeks later with 2 x 10(3) irradiated P. yoelii sporozoites, and were challenged several weeks later. The presence of PyHsp60 in the immunization regimen did not lead to reduced blood-stage infection or development of parasites in hepatocytes. PyHsp60 DNA vaccines were immunogenic in BALB/c mice but did not consistently, completely protect against sporozoite challenge. The observation that in some of the PyHsp60 DNA vaccine-immunized mice there was protection against infection or a delay in the onset of parasitemia after sporozoite challenge deserves further evaluation.

Functional genomic technologies applied to the control of the human malaria parasite, Plasmodium falciparum.

Infection with any of the four species of Plasmodium single cell parasites that infects humans causes the clinical disease, malaria. Of these, it is Plasmodium falciparum that is responsible for the majority of the 1.5-2.3 million deaths due to this disease each year. Worldwide there are between 300-500 million cases of malaria annually. To date there is no licensed vaccine and resistance to most of the available drugs used to prevent and/or treat malaria is spreading. There is therefore an urgent need to develop new and effective drugs and vaccines against this devastating parasite. We have outlined a strategy using a combination of DNA-based vaccines and the data derived from the soon-to-be completed P. falciparum genome and the genomes of other species of Plasmodium to develop new vaccines against malaria. Much of the technology that we are developing for vaccine target identification is directly applicable to the identification of potential targets for drug discovery. The publicly available genome sequence data also provides a means for researchers whose focus may not be primarily malaria to leverage their research on cancer, yeast biology and other research areas to the biological problems of malaria.

Genomic tools for gene and protein discovery in malaria: toward new vaccines.

Advances in malaria vaccine and drug development have been hindered in part by the complex multistage life cycle of the parasite, much of which is inaccessible to study, and by a large genome encoding over 5000 genes. Two human models of immunity to malaria, however, suggest that the development of an effective vaccine is within reach. We have outlined a strategy to identify the expression of hundreds to thousands of potential vaccine targets employing recently developed technologies for gene and protein expression. Combined with the exciting developments of malaria DNA vaccine technologies, these approaches form the basis for malaria subunit vaccines that may mimic the protective efficacy of our human model systems and provide the foundation for novel approaches to vaccine development for a range of pathogens.

Malaria research in the post-genomic era.

Within the next few years, the complete genomic sequences of Plasmodium falciparum, and potentially several other Plasmodium spp, will be available to researchers worldwide. These complete genomic sequence data are certain to provide the foundation for nearly all malaria research in the next decades, as discussed here by Dan Carucci.

Guanylyl cyclase activity associated with putative bifunctional integral membrane proteins in Plasmodium falciparum.

We report here that guanylyl cyclase activity is associated with two large integral membrane proteins (PfGCalpha and PfGCbeta) in the human malaria parasite Plasmodium falciparum. Unusually, the proteins appear to be bifunctional; their amino-terminal regions have strong similarity with P-type ATPases, and the sequence and structure of the carboxyl-terminal regions conform to that of G protein-dependent adenylyl cyclases, with two sets of six transmembrane sequences, each followed by a catalytic domain (C1 and C2). However, amino acids that are enzymatically important and present in the C2 domain of mammalian adenylyl cyclases are located in the C1 domain of the P. falciparum proteins and vice versa. In addition, certain key residues in these domains are more characteristic of guanylyl cyclases. Consistent with this, guanylyl cyclase activity was obtained following expression of the catalytic domains of PfGCbeta in Escherichia coli. In P. falciparum, expression of both genes was detectable in the sexual but not the asexual blood stages of the life cycle, and PfGCalpha was localized to the parasite/parasitophorous vacuole membrane region of gametocytes. The profound structural differences identified between mammalian and parasite guanylyl cyclases suggest that aspects of this signaling pathway may be mechanistically distinct.

Plasmodium yoelii: cloning and characterization of the gene encoding for the mitochondrial heat shock protein 60.

Heat shock proteins are a highly conserved group of proteins required for the correct folding, transport, and degradation of other proteins in vivo. The Hsp70, Hsp90, and Hsp60 families are among the most widely studied families. Hsp60 is found in eubacteria, mitochondria, and chloroplasts, where, in cooperation with Hsp10, it participates in protein folding and translocation of proteins to the organelles. We have cloned and characterized the Hsp60 gene of Plasmodium yoelii (PyHsp60). PyHsp60 is a single-copy gene, located on chromosome 9, 10, or 11. The PyHsp60 cDNA sequence showed an open reading frame of 1737 nucleotides that codes for a polypeptide of 579 amino acids, with 93% amino acid identity to Plasmodium-falciparum Hsp60 (PfHsp60). Cloning and sequencing of a genomic PCR clone showed the presence of a 201-bp intron, located 141 bp downstream of the ATG codon. A single, heat-inducible, 2.3-kb transcript was detected in Northern blots of RNA isolated from blood stage parasites. Mouse antisera raised against a DNA vaccine vector that expresses PyHsp60 recognized sporozoites and liver- and blood-stage parasites by indirect fluorescent antibody test (IFAT). By Western blot, these antisera reacted with the mycobacterial Hsp65 and recognized a protein of approximately 65 kDa in P. yoelii sporozoites and P. falciparum blood stages. These results show that PyHsp60 and PfHsp60 genes are homologous and that of the PyHsp60 gene encodes a heat-inducible, intracellular protein that is expressed in several of the developmental stages of P. yoelii.

A shotgun optical map of the entire Plasmodium falciparum genome.

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7-2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).

Optical mapping of Plasmodium falciparum chromosome 2.

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.