WO2000025728A2 - Sequence du chromosome 2 du parasite du paludisme plasmodium falciparum et proteines dudit chromosome utiles pour les vaccins antipaludiques et reactifs de diagnostic - Google Patents

Sequence du chromosome 2 du parasite du paludisme plasmodium falciparum et proteines dudit chromosome utiles pour les vaccins antipaludiques et reactifs de diagnostic Download PDF

Info

Publication number
WO2000025728A2
WO2000025728A2 PCT/US1999/026796 US9926796W WO0025728A2 WO 2000025728 A2 WO2000025728 A2 WO 2000025728A2 US 9926796 W US9926796 W US 9926796W WO 0025728 A2 WO0025728 A2 WO 0025728A2
Authority
WO
WIPO (PCT)
Prior art keywords
proteins
protein
plasmodium falciparum
chromosome
infection
Prior art date
Application number
PCT/US1999/026796
Other languages
English (en)
Other versions
WO2000025728A3 (fr
Inventor
Stephen Hoffman
Daniel Carucci
Malcolm Gardner
J. Craig Venter
Original Assignee
United States Government As Represented By The Secretary Of The Navy
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by United States Government As Represented By The Secretary Of The Navy filed Critical United States Government As Represented By The Secretary Of The Navy
Priority to AU18182/00A priority Critical patent/AU1818200A/en
Publication of WO2000025728A2 publication Critical patent/WO2000025728A2/fr
Publication of WO2000025728A3 publication Critical patent/WO2000025728A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Chromosome 2 Sequence of the Human Malaria Parasite Plasmodium falciparum and Proteins of said Chromosome Useful in Anti-Malarial Vaccines and Diagnostic Reagents
  • This invention relates to proteins of gene models contained within chromosome 2 (the second largest of 14 identified chromosomes) from the human malaria parasite, Plasmodium falciparum (clone 3D7) and related families of gene products from other clones or strains of Plasmodium falciparum. These proteins facilitate anti-malarial vaccine development, drug discovery and the development of new diagnostic reagents.
  • Malaria a disease caused by protozoan parasites of the genus Plasmodium, is one of the most important infectious diseases affecting human populations. Approximately 300-500 million people are infected annually, and 1.5- 2.7 million lives are lost to malaria each year, with most deaths occurring among children in sub-Saharan Africa (1). Of the 4 species that cause malaria in humans, P. falciparum is responsible for the most morbidity and mortality. Parasite resistance to drugs and mosquito resistance to insecticides have led to a resurgence of malaria in many parts of the world, and a pressing need for vaccines and new drugs. Identification of new targets for vaccine and drug development is dependent upon expansion of our understanding of parasite biology, a process hampered by the complexity of the parasite life cycle. Sequencing of the Plasmodium genome promises to circumvent many of these difficulties and rapidly increase our knowledge about these parasites.
  • the P. falciparum genome is approximately 30 Mb in size, has a base composition of 82% AT, and contains 14 chromosomes ranging from 0.65 to 3.4 Mb. Chromosomes from different wild isolates exhibit extensive size polymorphism. Mapping studies indicate that the chromosomes contain central domains that are conserved between isolates and polymorphic subtelomeric domains containing repeated sequences.
  • P. falciparum also contains two organellar genomes.
  • the mitochondrial genome is a 5.9 kb tandemly-repeated DNA, and a 35 kb circular DNA that encodes genes usually associated with plastid genomes is located within the apicoplast, an organelle of uncertain function in Plasmodium and the related parasite Toxoplasma (2).
  • Genomic information is being generated from entire organisms at a rapid pace.
  • simple methods can be used to identify the regions of the DNA that are responsible for the production of proteins.
  • the regions of the genome that are identified to make up the final gene structure are not easily defined.
  • the resulting gene sequence that encodes a protein is actually made from fragments of the genome sequence.
  • the regions that constitute a protein- encoding gene are known as "exons" and the regions within that are excised from the final gene are known as "introns”.
  • an object of this invention is the use of the identified proteins, through the predicted gene models derived from the genetic sequence of Plasmodium falciparum chromosome 2, to identify potential novel targets for anti-Plasmodial and antimalarial drugs vaccines and diagnostics.
  • Another object of this invention is the identification of novel biochemical pathways that regulate cellular biochemistry, including but not limited to drug resistance. These protein models can then be used to develop diagnostics for the early identification of drug resistant Plasmodium species.
  • Another objective of this invention is the identification through protein homologies of potential drug, vaccine and diagnostic targets from related species of Plasmodium.
  • Fig. 1 is a gene map of the P. falciparum chromosome 2.
  • Fig. 2 is DNA sequence of multiple alignment of the predicted 5'-3' exonuclease (PFB0180w) encoded in chromosome 2 with homologous bacterial exonuclease domains showing the large non-globular insert in Plasmodium.
  • Fig. 3 is multiple sequence alignment of rifins encoded on chromosome 2. The predicted coding regions were aligned with CLUSTALW (32) using the default settings.
  • the alternatives to identifying these particular proteins includes a gene-by-gene approach either by amplification of DNA sequences by means such as degenerate polymerase chain reaction, or by hybridization of genomic or expression libraries. Neither of these alternatives is desirable, as none will ensure the full length of the predicted protein.
  • An advantage to the identification of expression libraries is that the biological determined intron- exon boundaries can be determined accurately.
  • expression libraries can only be developed from particular stages of an organism's life cycle and in the case of Plasmodium parasites that means, in a practical sense only from blood stage parasites Therefore by this method it is certain that many protein sequences will be left unidentified
  • Chromosome 2 was sequenced using the shotgun sequencing approach used previously to sequence several microbial genomes (3), with modifications to compensate for the AT- ⁇ chness of P falciparum DNA (4) The most important modifications were extraction of DNA from agarose under high-salt conditions to prevent melting of the
  • the assembly software was also modified to minimize miss-assembly of AT- ⁇ ch sequences
  • Chromosome 2 of P falciparum (clone 3D7) is 945 kb in length and has an overall base composition of 80 2%
  • the chromosome contains a large central region encoding single-copy and several duplicated genes, subtelome ⁇ c regions containing va ⁇ ant antigen genes (var) (5), RIF-1 elements (repetitive interspersed family) (6) and other repeats, and typical eukaryotic telomeres (Fig 1)
  • Figure 1 Gene map of P falciparum chromosome 2 Predicted coding regions are shown on each strand
  • Exons of protein encoding genes are indicated by rectangles with lines linking rectangles representing introns
  • the single tRNAGlu gene is indicated by a cloverleaf structure
  • Genes are color-coded according to broad role categories as shown in the key Gene identification numbers correspond to those in Table 2
  • the letters CC, NG and TM followed by numerals indicate the number of predicted coiled-coil, non-globular, and transmembrane domains in the proteins, respectively
  • the terminal 19 kb portions of the chromosome are non-coding and exhibit 77% identity in opposite orientations
  • the left and right telomeres consist of tandem repeats of the sequence TT(TC)AGGG (7) totaling 1141 and 551 nt, respectively
  • the subtelome ⁇ c regions do not exhibit repeat oligomers until approximately 12-20 kb into the chromosome, where rep20 (8), a 21 bp tandem direct repeat found exclusively in these regions, occurs 134 and 96 times in the left and ⁇ ght ends of the chromosome, respectively
  • the sequence similarity observed between the subtelome ⁇ c regions supports previous suggestions that recombination between chromosome ends may be one mechanism by which genetic diversity is generated
  • a region with centromere functions could not be identified based on sequence simila ⁇ ty to S
  • centrome ⁇ c functions may be defined by higher-order DNA structures and chromatin-associated protem complexes (10)
  • tRNAGlu Two hundred nine protein-encoding genes and a gene for tRNAGlu (Fig 1, Table 1) were predicted (11) on chromosome 2, giving a gene density of one gene per 4 5 kb, a value between that observed in yeast (one gene per 2 kb) and C elegans (one gene per 7 kb) Of the 209 protein-encoding genes, 43% contain at least one intron This is an estimate because some introns may have been missed by the gene finding method Most spliced genes consist of two or three exons In terms of intron content and gene density, the Plasmodium genome, assessed by the analysis of the first completed chromosome sequence, appears to be intermediate between the condensed yeast genome and the mtron- ⁇ ch genomes of multicellular eukaryotes Table 1. Summary of features of P. falciparum chromosome 2 and comparison to S. cerevisiae chromosome 3. ND, not determined. tProtein structural features were predicted as described (1 1).
  • Integral membrane with multiple predicted transmembrane domains (%) 27 (13) 21 (12) Containing coiled-coil domains (%) 111 (53) 32(19) Containing other large compositionally biased regions with predicted non-globular structure ( ;%%)) 155 (75) 71 (41) Completely non-globular (%) 17(8) 6 (3.5) With detectable homologs in other species 87 (42) 145 (85)
  • the proteins encoded in chromosome 2 fall into 3 categories: i) 72 proteins (34%) are conserved in other genera and contain one or more distinct globular domains; ii) 47 proteins (23%) belong to families with identifiable structural features and in some cases, known functions; iii) 90 predicted proteins (43%) have no detectable homologs, although many contain structural features such as signal peptides and transmembrane domains. Homologs outside Plasmodium were detected for 87 (42%) of the 209 predicted proteins. This includes proteins in the 1st category, plus those proteins in the 2nd category that possess conserved domain(s) arranged in a manner unique to Plasmodium. The percentage of evolutionarily conserved proteins is about two-fold lower than found for other genomes, mainly because most of the remaining proteins were predicted to consist primarily of non-globular domains
  • PF# systematic name assigned according to a method adapted from S. cerevisiae (11). Description: Name, if known, and prominent features of the gene. Abbreviations are as follows: euk, eukaryotic; nt, nucleotide; 00, organellar origin; TP, transit peptide.
  • acyl-CoA synthetase Purines, pyrimidines, nucleosides, and nucleotides
  • PFB0890c pseudouridine synthet. (RsuA fam.); 1st euk. member (00) Translation and post-translational modification PFB0165w tRNA-Glu
  • VPS45-like protein STXBP/UNC-18/SECl family
  • PFB0320c member hesB fam. (poss. redox activity, 00,TP)
  • RT-PCR was performed on 11 non-globular domains and two genes encoding predominantly non-globular proteins using total blood stage RNA as template. In all cases, RT-PCR products were the same size as those amplified from genomic DNA, and the sequence of RT-PCR products matched genomic DNA sequence (14). Thus, it is likely that most, if not all, predicted non-globular domains in chromosome 2 genes are expressed.
  • a non-globular domain into a well-defined globular domain is seen in a protein containing a 5'-3' exonuclease (Fig. 2). Figure 2.
  • the aspartates involved in metal coordination are shown by red background and inverse type. Secondary structure elements derived from the crystal structure of Thermus aquaticus DNA polymerase (15) are shown above the alignment (H indicates a-helix, and E indicates extended conformation, or b-strand). 5'-3-exo_Aae is a stand alone exonuclease from Aquifex aeolicus, and the remaining bacterial sequences are the N-terminal domains of DNA polymerase I. Alignment of the Plasmodium sequence with 4 bacterial exonucleases revealed a 176-amino acid insertion in a region between a strand and helix in the 3-dimensional structure of this protein (15).
  • One such gene encodes 3-ketoacyl-ACP synthase III (FabH), which catalyzes the condensation of acetyl-CoA and malonyl-ACP in Type II (dissociated) fatty acid synthase systems.
  • Type II synthase systems are restricted to bacteria and the plastids of plants, confirming previous hypotheses that the Plasmodium apicoplast contains metabolic pathways distinct from those of the host (17, 18).
  • chromosome 2 a subunit of HSP70-type molecular chaperones and participate in protein folding and trafficking, complex assembly, organelle biogenesis, and initiation of translation (19).
  • Five proteins containing DnaJ domains are present on chromosome 2, which suggests multiple roles for this domain in the Plasmodium life cycle. Two of these consist primarily of the DnaJ domain, whereas three also contain a large non- globular domain.
  • proteins containing a DnaJ domain have been detected on other chromosomes, indicating that this is a large gene family in Plasmodium (20).
  • the ring-infected erythrocyte surface antigen a protein folding and trafficking, complex assembly, organelle biogenesis, and initiation of translation (19).
  • Five proteins containing DnaJ domains are present on chromosome 2, which suggests multiple roles for this domain in the Plasmodium life cycle. Two of these consist primarily of the DnaJ domain, whereas three also contain a large non- globular
  • Chromosome 2 contains 5 protein families that are unique to Plasmodium in terms of their distinct domain organization, although 3 of them contain domains conserved in other genera. The genes encoding the Plasmodium- specific families are primarily located near the ends of the chromosome. A single var gene was identified in each subtelomeric region.
  • the var genes encode large transmembrane proteins (PfEMPl) expressed in knobs on the surface of schizont- infected red cells.
  • PfEMP-1 proteins exhibit extensive sequence diversity, are clonally-variant, and are involved in antigenic variation, cytoadherence, and resetting (5).
  • 6 small ORFs were identified in the subtelomeric regions that had similarity to var sequences. Five of these resembled var exon II cDNAs or Pf ⁇ O.l sequences reported previously (22, 23).
  • the largest Plasmodium-specific family found on chromosome 2 encodes proteins that were dubbed rifins, after the RIF-1 repetitive element.
  • RIF-1 contained a 1 kb ORF but no initiation codon, was found on most chromosomes, and was transcribed in late blood stage parasites. The function of the RIF-1 element was unknown. Eighteen ORFs with similarity to RIF-1 were found in the subtelomeric regions of chromosome 2, centromeric to the var genes. Inspection of the sequence upstream of these ORFs revealed exons encoding signal peptides, indicating that the RIF-1 elements are actually genes consisting of 2 exons.
  • transmembrane proteins encode potential transmembrane proteins with predicted molecular weights of 27-35 kD with an extracellular domain containing conserved Cys residues which might participate in disulfide bonding, one or more transmembrane segments, and a short basic C-terminus that is intracellular.
  • the extracellular domain also contains a highly variable region (Fig. 3).
  • RT-PCR with schizont RNA showed that 1 of 6 rifin genes tested was transcribed.
  • Figure 3 Multiple sequence alignment of rifins encoded on chromosome 2. The predicted coding regions were aligned with CLUSTALW (32) using the default settings.
  • the alignment column shading is based on a 95% consensus, which is shown underneath the alignment; h indicates hydrophobic residues (A,C,F,I,L,M,V,W,Y; yellow background), p indicates polar residues (D,E,H,K,N,Q,R,S,T; red coloring), b indicates big residues (F,I,L,M,V,W,Y, K,R,Q,E; gray background), and "+" indicates positively charged residues (K,R; red coloring).
  • the cysteines conserved in subsets of rifins are shown by blue shading and inverse coloring.
  • the function of the rifins is unknown, but their sequence diversity, predicted cell surface localization, and expression in erythrocytic stages suggests that like var genes they may be clonally-variant. In addition, it is reasonable to expect that because they are predicted to be expressed on the red cell surface, the rifins interact with host ligands and are involved in cytoadherence, resetting, or other pathogenic processes involving host- parasite interactions. This view is supported by others who suggest that the close proximity of the Rifin family of gene to the highly variable var genes means that the rifins encode for variable molecules that are present on the surface of the infected red cell (6a). Thus the rifins can reasonably be expected to be useful as targets of vaccine-induced immunity.
  • SERAs SE ⁇ ne Repeat Antigens
  • SERAs SE ⁇ ne Repeat Antigens
  • SERAs SE ⁇ ne Repeat Antigens
  • SERA genes are part of an 8-gene cluster, 7 genes have a similar 4 exon structure, but the gene at the 3' end of the cluster contains only 3 exons
  • the protease domains in these proteins are unusual in that 5 of the 8 contain se ⁇ ne instead of cysteine in the active nucleophile position, suggesting that they are se ⁇ ne proteases with a structure typical of cysteine proteases (27)
  • MSP-4 and MSP-5 which contain an epidermal growth factor (EGF) module m their extracellular domains (28, 29) Together with MSP-1, a multi-EGF domain protein encoded on chromosome 3, and two Plasmodium sexual stage antigens (30), these are the only proteins outside the animal kingdom that contain EGF repeats, suggesting that the sequence for this domain was obtained by Plasmodium from its animal host The plasmodial EGF domains may be involved in parasite adhesion to host cells
  • chromosome 2 contains genes for many secreted and membrane proteins
  • PFB0570w encodes a protein with a modified thrombospondin domain, and was transc ⁇ bed in blood stage parasites
  • Other Plasmodium proteins containing thrombospondin domains such as sporozoite surface protein 2/TRAP and circumsporozoite protein, are involved in parasite invasion of host cells (31), and it is reasonable to expect that the protein encoded by gene PFB0570w is involved m the binding of infected red cells or extracellular parasites to host cell ligands
  • the protein encoded by PFB0570w can reasonably be expected to be useful as a target of vaccine-induced immunity
  • Interference of the parasite-host ⁇ nteract ⁇ on(s) mediated by PFB0570w by the use of drugs that inhibit or prevent such mteract ⁇ on(s) can also be expected to have therapeutic benefits.
  • Plasmodium genes encoding parasite transmembrane or surface proteins can be inserted into mammalian expresssion vectors in order to construct DNA vaccines, that when used to immunize experimental animals, induce humoral and cellular immune responses to the Plasmodium protein(s), including protective cytotoxic T cell responses;
  • a DNA vaccine encoding P.falciparum circumsporozoite protein has also been shown to induce cytotoxic T cell responses in human volunteers (31i).
  • the predicted transmembrane and secreted proteins encoded on chromosome 2 can also be expected to induce protective cellular or humoral immune responses when formulated as DNA vaccines or as other types of vaccines such as recombinant proteins. Portions or fragments of the proteins that contain B or T cell epitopes encoded on chromosome 2 may also be used in the construction of vaccines (31 e, 3 lj).
  • Determination of the first P.falciparum chromosome sequence demonstrates that the AT-richness of P.falciparum DNA will not prevent sequencing of the genome. Although technical difficulties not observed during the sequencing of other microbial genomes were encountered, solutions to these problems were found that will facilitate sequencing of the remaining chromosomes.
  • the genome sequence will be of great value in the study of Plasmodium biology, and the development of new drugs and vaccines for the treatment and prevention of malaria. In addition to these practical benefits, the Plasmodium genome sequence will provide broader biological insights, particularly with regard to the plasticity of the eukaryotic genome manifest in the preponderance of the predicted non-globular domains in plasmodial proteins.
  • Example 1 The genes that encode the family of Rifins will be amplified using the polymerase chain reaction and cloned into DNA vaccines - a plasmid vector designed to expressed the cloned fragment when injected into human or animal tissue. These Rifin vaccines will express the individual Rifin polypeptide using cellular protein expression systems. Each DNA vaccine will be designed to express an individual Rifin polypeptide. These polypeptides will then be taken up by antigen presenting cells and the host immune system will respond by producing either cellular or humoral immune responses directed at each of the expressed polypeptides.
  • This immune response will prevent or reduce Plasmodium falciparum parasite development in host cells by preventing the invasion of parasites into erythrocytes, or by opsonization which will result in the clearance of parasites by antibody mediated cellular immunity.
  • These antibodies may act by blocking merozoite invasion of erythrocytes by blocking the initial interaction with the erythrocyte cell surface agglutinate merozoites before invasion or at, or immediately preceding, rupture of the mature schizont.; by killing the infected erythrocyte, via either complement-mediated lysis or phagocytosis; by preventing mature schizonts from adhering to endothelial cells (cytoadherence) by blocking receptor/ligand interactions, thereby preventing sequestration and enhancing splenic clearance; by preventing the release of, or inactivate, harmful toxins released from the infected erythrocyte; by antibody dependent or antibody independent cellular mechanisms directed against the merozoite in circulation or the intracellular parasite
  • Example 2 The genes involved in critical biochemical pathways, such as fatty acid biosynthesis can be identified as targets for antimalarial drug development.
  • the identified polypeptides will be expressed as recombinant proteins and used in in vitro drug screening assays to identify chemical compounds that interfere with the normal functioning of the recombinant polypeptide. These drugs will then be taken through traditional drug development and to clinical trials for efficacy.
  • Example 3 The genes identified in chromosome 2 can be used to produce diagnostic reagents for laboratory or field detection of P.falciparum parasites. Using high throughput methods, such as DNA microarray technology or mass spectrometry , or other techniques, the genes and/or proteins that are identified to be expressed in abundance can be used to develop diagnostic reagents. For example, DNA probes can be designed which will hybridize to parasite nucleic acid extracts and then be useful to determine the presence of parasites in a clinical sample. Identified proteins that are shown to be highly expressed in blood stage parasites will be used to produce recombinant proteins used to produce antisera in experimental animals. These sera will then be used in diagnostic assays to detect parasites in clinical samples. Example 4.
  • DNA probes can be designed which will hybridize to parasite nucleic acid extracts and then be useful to determine the presence of parasites in a clinical sample.
  • Identified proteins that are shown to be highly expressed in blood stage parasites will be used to produce re
  • genes identified in chromosome 2 will be used in high throughput assays to identify those that are involved in the development of anti malarial drug resistance. These genes can then be exploited as in Example 3, in the development of drug resistant diagnostic tests. For example, antisera against the identified drug resistance proteins can be used in an ELISA assay to detect the expression of proteins in parasite extracts or clinical samples that are involved in parasite-mediated drug resistance.
  • Chromosomes were resolved on preparative pulsed field gels (1.2% SeaPlaque GTG agarose, BioRad DRI ⁇ apparatus, 180-250 sec switch time, 120 field angle, 3.7 V/cm for 90 hours at 14DC). Chromosome 2 bands from 5 gels were adjusted to 0.3 M sodium acetate to prevent melting of the AT-rich DNA and digested with agarase. Exposure to
  • a shotgun library of 1-2 kb fragments was prepared in pUC18 as described [R. D. Fleischmann, et al., Science 269, 496-512 (1995)], except that treatment with E. coli DNA polymerase I was performed (0.5 mM dNTPs, 37DC for 10 minutes) after the second ligation step to close nicks prior to electropor ation into DH10B cells. Because the gel-purified chromosome 2 DNA was only -85% pure due to co-migration of sheared DNA from other chromosomes, and to provide excess coverage to compensate for possible non-randomness of the shotgun library,
  • Cycling conditions Perkin Elmer GeneAmp PCR Systems 9600 or 9700 were 94DC for 2 min, followed by 10 cycles of 94DC for 1 min, 50 or 55DC for 1 min, and 60OC for 2 min, 20 cycles of 94DC for 1 min, 50 or 55DC for 1 min, and 60DC for 2 min plus 20 sec per cycle, and 1 cycle at 60GC for 10 min.
  • PCR products were purified (QIAquick PCR Purification Kit; Qiagen 28104) and sequenced using dye-terminator chemistry. Sequence gaps that were too AT-rich for primer synthesis and walking were closed by insertion of the artificial transposon AT-2 [S. E. Devine, J. D. Boeke, Nucleic Acids Res.
  • P.falciparum clone 3D7 was selected because it can complete all stages of the life cycle, and was used in a genetic cross [D. Walliker, I. Quayki, T. E. Wellems, T. F. McCutchan, Science 236, 1661-1666 (1987)], and the Wellcome Trust Malaria Genome Mapping Project [J. Foster, J. Thompson, Parasitol. Today 11, 1-4 (1995)]. Parasites were grown in vitro [W. Trager, W. Jensen, Nature 273, 621-622 (1978)] and embedded in agarose [D. J. Kemp, et al., Nature 315, 347-50 (1985)]. Chromosomes were resolved on preparative pulsed field gels (1.2% SeaPlaque GTG agarose, BioRad DRIII apparatus, 180-250 sec switch time, 120 field angle, 3.7 V/cm for 90 hours at 14DC).
  • Chromosome 2 bands from 5 gels were adjusted to 0.3 M sodium acetate to prevent melting of the AT-rich DNA and digested with agarase. Exposure to UV light was minimized.
  • a shotgun library of 1-2 kb fragments was prepared in pUC18 as described [R. D. Fleischmann, et al., Science 269, 496-512 (1995)], except that treatment with E. coli DNA polymerase I was performed (0.5 mM dNTPs, 373 C for 10 minutes) after the second ligation step to close nicks prior to electroporation into DH10B cells.
  • PCR reactions with genomic DNA template were done with primers from adjacent mapped groups, or from one mapped group and each of the unmapped groups.
  • PCR reactions (Expand Long Template PCR System, Boerhinger Mannheim 1681 842) contained 100 ng of genomic DNA and 15 pmol of each primer (BioServe Technologies) in a 50 ml reaction.
  • Cycling conditions Perkin Elmer GeneAmp PCR Systems 9600 or 9700 were 94 DC for 2 min, followed by 10 cycles of 94DC for 1 min, 50 or 55DC for 1 min, and 60DC for 2 min, 20 cycles of 94DC for 1 min, 50 or 55DC for 1 min, and 60DC for 2 min plus 20 sec per cycle, and 1 cycle at 60GC for 10 min.
  • PCR products were purified (QIAquick PCR Purification Kit; Qiagen 28104) and sequenced using dye-terminator chemistry. Sequence gaps that were too AT-rich for primer synthesis and walking were closed by insertion of the artificial transposon AT-2 [S. E. Devine, J. D. Boeke, Nucleic Acids Res.
  • NR non-redundant protein sequence database at the National Center for Biotechnology Information (NIH, Bethesda) was searched using the gapped BLAST and PSI-BLAST programs. Coding regions were predicted using GlimmerM [S. L. Salzberg, M. Pertea, A. Delcher, M. J. Gardner, H. Tettelin, Genomics 59, 24-31 (1999)], a eukaryotic gene-finding program based on Glimmer [S. L. Salzberg, A. L. Delcher, S. Kasif, O. White, Nucleic
  • Transfer RNAs were identified with tRNAscan [T. M. Lowe, S. R. Eddy, Nucleic Acids Res 25, 955-64 (1997)].
  • Systematic gene names based on a scheme used for S. cereviseae [H. W. Mewes, et al., Nature 387, 7-65 (1997)] were assigned using the convention PF (for P.falciparum), a letter for the chromosome (A for chromosome 1, B for chromosome 2, etc.), a 3-digit code ordering the genes from left-to-right in increments of 5 (to allow for addition of new genes), and a letter denoting the coding strand (w or c).
  • non-globular refers to proteins or domains of proteins that do not assume compact, folded structures [J. C. Wootton, Comput Chem 18, 269-85 (1994)].
  • compositional bias in protein sequences There is a strong inverse correlation between compositional bias in protein sequences and their ability to fold into a compact, globular domain [J. C. Wootton, S. Federhen, Methods Enzymol 266, 554-571 (1996)].
  • the compositional complexity of a sequence can be used to partition it into predicted globular and non-globular domains. In this analysis, this prediction was performed using the SEG program with the following parameters: window length 45, trigger complexity 3.4, extension complexity 3.75.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Le séquençage du chromosome 2 du Plasmodium falciparum a démontré que ce dernier contenait 945.000 paires de base et codait pour 209 gènes prévus. Comparé au génome de Saccharomyces cerevisiae, le chromosome 2 présente une densité génétique inférieure, les introns sont plus fréquents et les protéines sont enrichies de façon significative dans les domaines non globulaires. Une nouvelle famille de protéines de surface, les rifines, ont été identifiées. On suppose que les rifines jouent un rôle dans la variation antigénique. La séquence de génomes jette les bases du développement de méthodes de lutte contre le paludisme, une maladie qui tue des millions de personnes chaque année.
PCT/US1999/026796 1998-11-05 1999-11-05 Sequence du chromosome 2 du parasite du paludisme plasmodium falciparum et proteines dudit chromosome utiles pour les vaccins antipaludiques et reactifs de diagnostic WO2000025728A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU18182/00A AU1818200A (en) 1998-11-05 1999-11-05 Chromosome 2 sequence of the human malaria parasite (plasmodium falciparum) and proteins of said chromosome useful in anti-malarial vaccines and diagnostic reagents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10713198P 1998-11-05 1998-11-05
US60/107,131 1998-11-05

Publications (2)

Publication Number Publication Date
WO2000025728A2 true WO2000025728A2 (fr) 2000-05-11
WO2000025728A3 WO2000025728A3 (fr) 2001-02-22

Family

ID=22315022

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/026796 WO2000025728A2 (fr) 1998-11-05 1999-11-05 Sequence du chromosome 2 du parasite du paludisme plasmodium falciparum et proteines dudit chromosome utiles pour les vaccins antipaludiques et reactifs de diagnostic

Country Status (2)

Country Link
AU (1) AU1818200A (fr)
WO (1) WO2000025728A2 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092628A2 (fr) * 2001-05-16 2002-11-21 Institut Pasteur Antigenes de plasmodium falciparum et leurs applications vaccinales et diagnostiques
WO2004067559A1 (fr) * 2003-01-27 2004-08-12 Københavns Universitet Composes utilises pour le diagnostic et la prevention de la malaria associee a la grossesse
WO2007060550A3 (fr) * 2005-11-23 2008-01-03 Pasteur Institut Protéines de surface 4 et 5 du mérozoïte de plasmodium falciparum recombinées et utilisation de celles-ci
US7419672B2 (en) 1998-12-22 2008-09-02 Emergent Product Development Uk Limited Genes and proteins, and their use
US7592011B2 (en) 1998-12-22 2009-09-22 Emergent Product Development Uk Limited Genes and proteins, and their use
US8017745B2 (en) * 2002-12-06 2011-09-13 Epimmune Inc. Plasmodium falciparum antigens and methods of use
US20120244178A1 (en) * 2011-03-25 2012-09-27 Denise Doolan Plasmodium falciparum antigens
WO2016207408A1 (fr) * 2015-06-26 2016-12-29 Institute For Research In Biomedicine Nouveaux vaccins pour la prévention et le traitement de la malaria
WO2018215612A1 (fr) * 2017-05-24 2018-11-29 Ekkehard Werner Vaccin de plasmodium falciparum et de plasmodium vivax
CN112592395A (zh) * 2020-12-11 2021-04-02 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) 一种恶性疟原虫RIFIN重组蛋白PfRIFIN-54的构建、制备及用途

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017187A1 (fr) * 1993-01-29 1994-08-04 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services CLONAGE ET EXPRESSION DE L'ANTIGENE CIBLE BLOQUANT LA TRANSMISSION DE PLASMODIUM FALCIPARUM, Pfs230

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994017187A1 (fr) * 1993-01-29 1994-08-04 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services CLONAGE ET EXPRESSION DE L'ANTIGENE CIBLE BLOQUANT LA TRANSMISSION DE PLASMODIUM FALCIPARUM, Pfs230
US5733772A (en) * 1993-01-29 1998-03-31 The United States Of America As Represented By The Department Of Health And Human Services Cloning and expression of Plasmodium falciparum transmission blocking target antigen, Pfs230

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WILLIAMSON K.C. ET AL.: 'Cloning and expression of the gene for Plasmodium falciparum transmission-blocking target antigen, Pfs230' MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 58, 1993, pages 355 - 358, XP002930511 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7419672B2 (en) 1998-12-22 2008-09-02 Emergent Product Development Uk Limited Genes and proteins, and their use
US7592011B2 (en) 1998-12-22 2009-09-22 Emergent Product Development Uk Limited Genes and proteins, and their use
WO2002092628A2 (fr) * 2001-05-16 2002-11-21 Institut Pasteur Antigenes de plasmodium falciparum et leurs applications vaccinales et diagnostiques
WO2002092628A3 (fr) * 2001-05-16 2003-09-25 Pasteur Institut Antigenes de plasmodium falciparum et leurs applications vaccinales et diagnostiques
US7498037B2 (en) 2001-05-16 2009-03-03 Institut Pasteur Plasmodium falciparum antigens and their vaccine and diagnostic applications
US8017745B2 (en) * 2002-12-06 2011-09-13 Epimmune Inc. Plasmodium falciparum antigens and methods of use
WO2004067559A1 (fr) * 2003-01-27 2004-08-12 Københavns Universitet Composes utilises pour le diagnostic et la prevention de la malaria associee a la grossesse
US7745580B2 (en) 2003-01-27 2010-06-29 Kobenhavns Universitet Compounds useful in the diagnosis and treatment of pregnancy-associated malaria
EP2329844A1 (fr) * 2005-11-23 2011-06-08 Institut Pasteur Protéines de surface 4 et 5 du mérozoïte de plasmodium falciparum recombinées et utilisation de celles-ci
WO2007060550A3 (fr) * 2005-11-23 2008-01-03 Pasteur Institut Protéines de surface 4 et 5 du mérozoïte de plasmodium falciparum recombinées et utilisation de celles-ci
US8026354B2 (en) * 2005-11-23 2011-09-27 Institut Pasteur Recombinant plasmodium falciparum merozoite surface proteins 4 and 5 and their use
US8350019B2 (en) 2005-11-23 2013-01-08 Institut Pasteur Recombinant Plasmodium falciparum merozoite surface proteins 4 and 5 and their use
US8378087B2 (en) 2005-11-23 2013-02-19 Institut Pasteur Recombinant Plasmodium falciparum merozoite surface proteins 4 and 5 and their use
US20120244178A1 (en) * 2011-03-25 2012-09-27 Denise Doolan Plasmodium falciparum antigens
WO2016207408A1 (fr) * 2015-06-26 2016-12-29 Institute For Research In Biomedicine Nouveaux vaccins pour la prévention et le traitement de la malaria
WO2018215612A1 (fr) * 2017-05-24 2018-11-29 Ekkehard Werner Vaccin de plasmodium falciparum et de plasmodium vivax
CN112592395A (zh) * 2020-12-11 2021-04-02 中国疾病预防控制中心寄生虫病预防控制所(国家热带病研究中心) 一种恶性疟原虫RIFIN重组蛋白PfRIFIN-54的构建、制备及用途

Also Published As

Publication number Publication date
AU1818200A (en) 2000-05-22
WO2000025728A3 (fr) 2001-02-22

Similar Documents

Publication Publication Date Title
Gardner et al. Chromosome 2 sequence of the human malaria parasite Plasmodium falciparum
Doolan et al. Profiling humoral immune responses to P. falciparum infection with protein microarrays
Liu et al. Gametocytogenesis in malaria parasite: commitment, development and regulation
Bautista et al. Malaria proteomics: insights into the parasite–host interactions in the pathogenic space
WO2000025728A2 (fr) Sequence du chromosome 2 du parasite du paludisme plasmodium falciparum et proteines dudit chromosome utiles pour les vaccins antipaludiques et reactifs de diagnostic
Blair et al. Plasmodium falciparum MAEBL is a unique member of the ebl family
Karcz et al. Cloning and characterization of a vacuolar ATPase A subunit homologue from Plasmodium falciparum
Gardiner et al. Implication of a Plasmodium falciparum gene in the switch between asexual reproduction and gametocytogenesis
Chenik et al. Approaches for the identification of potential excreted/secreted proteins of Leishmania major parasites
Blake et al. Parasite genetics and the immune host: recombination between antigenic types of Eimeria maxima as an entree to the identification of protective antigens
Paoletta et al. The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell
Shuaibu et al. Selection and identification of malaria vaccine target molecule using bioinformatics and DNA vaccination
Sanchez et al. Plasmodium yoelii: cloning and characterization of the gene encoding for the mitochondrial heat shock protein 60
Black et al. MSP8 is a non-essential merozoite surface protein in Plasmodium falciparum
EP2601211B1 (fr) Vaccin
Nogueira et al. Plasmodium falciparum multidrug resistance protein (MRP) gene expression under chloroquine and mefloquine challenge
García et al. Well-defined regions of the Plasmodium falciparum reticulocyte binding protein homologue 4 mediate interaction with red blood cell membrane
Ajibaye Polymorphisms in Plasmodium falciparummalaria PfAMA1 and PfRH5 Genes in Isolates from Selected Centres in Lagos, Nigeria
Modrzynska Genetics of drug resistance in malaria: identification of genes conferring chloroquine and artemisinin resistance in rodent malaria parasite Plasmodium chabaudi
Young Plasmodium falciparum functional genomics: Elucidation of gene function and transcriptional control mechanisms
Mira Syahfriena Genetic diversity and natural selection of the rhoptry-associated protein 1 (RAP-1) of recent Plasmodium knowlesi clinical isolates from Malaysia/Mira Syahfriena Amir Rawa
Oyebola Genome-wide Analysis and Diversity of Plasmodium Falciparum in Lagos and Ekiti States, South-West Nigeria
Mwikali RNA sequencing analysis of Plasmodium falciparum extracellular vesicles
Birkholtz Molecular characterisation of the ornithine decarboxylase gene of the human malaria parasite, Plasmodium falciparum
Fox Regulation of TamS1 gene expression during differentiation to the merozoite in Theileria annulata

Legal Events

Date Code Title Description
ENP Entry into the national phase

Ref country code: AU

Ref document number: 2000 18182

Kind code of ref document: A

Format of ref document f/p: F

AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase