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EP0217877A1 - Antigenes hautement repetitifs de plasmodium falciparum - Google Patents

Antigenes hautement repetitifs de plasmodium falciparum

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Publication number
EP0217877A1
EP0217877A1 EP86902288A EP86902288A EP0217877A1 EP 0217877 A1 EP0217877 A1 EP 0217877A1 EP 86902288 A EP86902288 A EP 86902288A EP 86902288 A EP86902288 A EP 86902288A EP 0217877 A1 EP0217877 A1 EP 0217877A1
Authority
EP
European Patent Office
Prior art keywords
antigen
falciparum
polypeptide
arp
mesa
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP86902288A
Other languages
German (de)
English (en)
Other versions
EP0217877A4 (fr
Inventor
David James Kemp
Robin Fredric Anders
Graham Vallancey Brown
Ross Leon Coppel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Walter and Eliza Hall Institute of Medical Research
Original Assignee
Walter and Eliza Hall Institute of Medical Research
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Publication date
Application filed by Walter and Eliza Hall Institute of Medical Research filed Critical Walter and Eliza Hall Institute of Medical Research
Publication of EP0217877A1 publication Critical patent/EP0217877A1/fr
Publication of EP0217877A4 publication Critical patent/EP0217877A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • 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

  • This invention relates to the identification and production of highly repetitive antigens of Plasmodium falciparum and to DNA molecules comprising artificially constructed polynucleotide sequence ⁇ substantially corresponding to all or a portion of the base sequence coding for these antigens.
  • P.falciparum polypeptides are natural immunogen ⁇ in man but it i ⁇ by no means clear how many are important in protective immunity. Many antigens may have no such role, and indeed it is 5 possible that some are counterproductive, perhaps because collectively they overload the immune system. Antigenic diversity among different strains of the parasite may also play a significant role in the process of immune evasion as a number of P.falciparum antigens Q that are strain- ⁇ pecific have been identified.
  • the present invention is based upon the discovery of further antigens from P.falciparum, here designated the Small Histidine-Alanine Rich Protein (SHARP) - formerly called the 21K-Histidine Rich Protein (21K-HRP), the Asparagine-Rich Protein (ARP), and the Mature-parasite-infected Erythrocyte Surface Antigen (MESA) - formerly called the Glutami ⁇ Acid Rich Protein (GARP) , respectively, which are each characterized by a relatively high content of a single amino acid.
  • SHARP Small Histidine-Alanine Rich Protein
  • 21K-HRP 21K-Histidine Rich Protein
  • ARP Asparagine-Rich Protein
  • MEA Mature-parasite-infected Erythrocyte Surface Antigen
  • GARP Glutami ⁇ Acid Rich Protein
  • P.falciparum Human antibodie ⁇ affinity-purified on immobilized ly ⁇ ate ⁇ of the SHARP cDNA clone identified the corre ⁇ ponding para ⁇ ite antigen (in P.falciparum i ⁇ olate FCQ-27/PNG) a ⁇ a polypeptide of approximate Mr 29,000; affinity-purified human antibodie ⁇ to the ARP clone reacted with ⁇ everal para ⁇ ite polypeptide ⁇ in immunoblots, including antigens of approximate Mr 220,000 and 160,000, and human antibodies affinity-purified on immobilized lysate ⁇ of the MESA cDNA clone identified the corre ⁇ ponding parasite antigen as a polypeptide of Mr ⁇ .250,000.
  • a DNA molecule comprising a nucleotide sequence substantially corresponding to all or a portion of the base sequence coding for the Small Histidine-Alanine Rich Protein (SHARP) , the A ⁇ paragine-Rich Protein (ARP) , or the Mature-parasite-infected Erythrocyte Surface Antigen (MESA) of P.falciparum.
  • SHARP Small Histidine-Alanine Rich Protein
  • ARP A ⁇ paragine-Rich Protein
  • MESA Mature-parasite-infected Erythrocyte Surface Antigen
  • a DNA molecule comprising a nucleotide sequence characterized by at least a portion thereof comprising all or a portion of the base sequence shown in Figure 2, Figure 8 or Figure 11.
  • Such a nucleotide sequence codes for a polypeptide comprising at least a portion which corresponds to a portion of the amin ⁇ acid sequence of SHARP, ARP, or MESA, respectively.
  • the present invention also extends to synthetic peptides or polypeptides di ⁇ playing the antigenicity of all or a portion of SHARP, ARP, " or MESA, a ⁇ well as to compositions for stimulating immune respon ⁇ e ⁇ again ⁇ t SHARP, ARP, or MESA in a mammal, which compo ⁇ itions comprise at least one synthetic polypeptide displaying the antigenicity of all or a portion of SHARP, ARP, or MESA, respectively, together with a pharmaceutically acceptable carrier therefor.
  • the synthetic peptides or polypeptides according to this aspect of the invention may be prepared by expression in a host cell containing a recombinant DNA molecule which comprise ⁇ a nucleotide sequence as broadly described above operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule.
  • the synthetic peptide or polypeptide so expres ⁇ ed may be a fu ⁇ ion polypeptide compri ⁇ ing in addition to a portion di ⁇ playing the antigenicity of all or a portion of SHARP, ARP, or MESA an additional polypeptide coded for by the DNA of the recombinant DNA molecule.
  • the ⁇ ynthetic peptide ⁇ or polypeptides may be produced by chemical means, such as by the well-known Merrifield solid-phase synthe ⁇ is procedure.
  • Figure 1 shows the detection of the P.falciparum polypeptide corresponding to clone Ag57.
  • Proteins in a sonicated extract of clone Ag57 were conjugated to CNBr- activated Sepharose. The resulting conjugate was used as an absorbent for affinity purification of anti-Ag57 antibodies from a pool of human sera collected from individuals from Madang, Papua New Guinea.
  • Protein extracts of cultures of P.falciparum isolate ⁇ NF7(1), Kl (2), FC27(3), VI(4), and the cloned line ⁇ D10(5) derived from FC27 were fractionated on 10% polyacrylamide-SDS- gel ⁇ .
  • Protein ⁇ * from the gel ⁇ were tran ⁇ ferred electrophoretically to nitrocellulo ⁇ e, and detected by autoradiography after reaction with the affinity-purified antibodie ⁇ followed by 125I-protein A.
  • the antigen corre ⁇ ponding to clone Ag57 was pre ⁇ ent in each isolate (Mr range 29,000-34,000).
  • the antibodies cross reacted with a larger antigen (Mr range approximately 50,000-60,000) which was absent from
  • Figure 2 show ⁇ the DNA ⁇ equence and deduced amino acid sequence of Ag57.
  • the dideoxy chain termination method (9) was employed for sequence determination.
  • the insert of Ag57 and fragments generated from it by digestion with Ahalll and Rsal were cloned into M13mp8 and 9.
  • the complete sequence was determined in both directions, using the M13 "universal" primer on specific oligonucleotides where appropriate.
  • the propo ⁇ ed initiation site and repeated sequences are underlined.
  • Figure 3 shows a diagonal comparison of SHARP sequence ⁇ , compo ⁇ ed using the "Diagon" program of Staden (10) .
  • Figure 4 shows the detection of Ag319 by colony immunoassay. Human antibodies were reacted with the array of 103 antigen-positive clones derived from an expression library of NF7.
  • Figure 5 shows immunoblots of ⁇ upernatants from saponin-ly ⁇ ed infected erythrocyte ⁇ ; with human antibodies purified on Ag319.
  • Figure 6 show ⁇ :
  • Figure 7 ⁇ how ⁇ the ⁇ tructure of the cDNA clone Ag319 and the ⁇ trategy for determining it ⁇ ⁇ equence.
  • the repeats are indicated by filled triangles.
  • Arrows below represent the extent of sequencing runs from Ahalll (abbreviated A) , Rsal-site ⁇ and the linker.
  • the arrow with an a ⁇ terisk show ⁇ a run obtained with a ⁇ ynthetic primer corre ⁇ ponding to the complementary sequences from position 714-735 of Ag319.
  • Figure 8 shows the nucleotide and deduced amino acid sequence of Ag319.
  • Figure 9 is a protein "Diagon" according to the program of Staden (10) .
  • the deduced protein sequence of Ag319 was compared to itself.
  • the octapeptide repeats are clearly showing up as well as multiple internal homologies due to the interspersed Asn-rich tetrapeptides.
  • Figure 10 shows immunoblots with human antibodies affinity-purified from plasma pooled from individuals exposed to malaria.
  • A Equivalent numbers of parasitized erythrocytes containing synchronous stages of FC27 were harvested from culture at specific times during one cycle of growth. The parasites were collected by centrifugation and washed in serum-free medium. The cells were solubilized in PBS containing 0.5% Triton X100 and protea ⁇ e inhibitors (PMSF, 5mM; TPCK, lmK; EDTA, 2.5mM and iodoacetamide, 2mM) . The supernatant ⁇ were diluted 1:4 in SDS ⁇ ample buffer containing 2ME and boiled for 2 min. The polypeptide ⁇ were fractionated by PAGE, electroblotted to nitrocellulo ⁇ e and probed with anti-Ag7 antibodie ⁇ followed by 125I protein A.
  • PMSF Triton X100 and protea ⁇ e inhibitors
  • Figure 11 shows the nucleotide sequence and predicted amino acid sequence of Ag7.
  • the nucleotide sequence was determined by the dideoxy chain-termination procedure (9) and translated using the DBUTIL programs of Staden (10) .
  • the repeats are indicated by vertical bars, and the start and end points of Ag652 and 653 are indicated by arrows. Regions outside the repeat containing short regions of related sequence are underlined.
  • Figure 12 show ⁇ hybridization of Ag7 cDNA to restriction fragments of P.falciparum DNA, and to P.falciparum chromosome ⁇ .
  • the i ⁇ olate ⁇ were FC27 from Papua New Guinea (track ⁇ 1 and 4) Kl from Thailand (track ⁇ 2 and 6) and NF7 from Ghana (track ⁇ 3 and 5) .
  • Chromo ⁇ ome ⁇ from P.falciparum isolates were prepared and fractionated by pulsed-field gradient gel electrophoresis a ⁇ described (11) , blotted to nitrocellulose and hybridized as above.
  • the isolate ⁇ were clone E12, derived from FC27 (track 1), NF7 (track 2) and Kl (track 3).
  • Figure 13 ⁇ hows indirect immunofluorescence of P.falciparum asexual blood stages reacted with human antibodies to Ag651. Fluorescein ⁇ taining of ring (R) and trophozoite (T) stage para ⁇ ite ⁇ in an acetone-fixed ⁇ mear of isolate VI (panel A) or FC27 (panel B) . The in ⁇ erts are of mature schizonts.
  • Figure 14 is an immunoelectromicrograph showing a section of an erythrocyte infected with a mature stage P.falciparum parasite that had been reacted with human anti Ag7. Magnification is X35,600.
  • P.falciparum isolate FCQ27/PNG(FC27) was obtained through collaboration with the Papua New Guinea Insitute of Medical Re ⁇ earch. NF7, originating from Ghana, and Kl from Thailand were obtained from D.Walliker, Edinburgh University. Parasite ⁇ were maintained in asynchronou ⁇ culture as de ⁇ cribed by Trager and Jen ⁇ en (12) . Synchroni ⁇ ation of growth to within a 6hr ⁇ pread of maturation wa ⁇ achieved with two round ⁇ of ⁇ orbitol treatment (13) .
  • erythrocytes were harvested during one cycle of growth at 4, 26 and 38 hours after the second sorbitol treatment to obtain ring- ⁇ tage (>99%) , trophozoite (>97%) and schizont (>98%) preparations.
  • Merozoites naturally relea ⁇ ed over a 2hr period were purified from culture supernatants as described by Mrema et al (14) .
  • Construction of P.falciparum cDNA clones from FC27 mRNA, cloned in ⁇ gt/ll-Amp3 ( Amp3) was described (1) .
  • Sera were obtained with informed con ⁇ ent from individual ⁇ from Madang, Papua New Guinea through Dr.M.P.Alper ⁇ and a ⁇ ociate ⁇ , Papua New Guinea In ⁇ titute of Medical Research. 10
  • Replicas of antigen-positive clones were grown overnight at 30°C, induced at 42°C, and lysed (1) . Sera were absorbed to remove anti-E.coli reactivity, diluted
  • Induced 50ml cultures of antigen-positive clones were prepared as described previously (17) .
  • the pelleted bacteria were sonicated and soluble bacterial proteins were conjugated to CNBr-activated Sepharose (Pharmacia, Sweden).
  • Antibodies from a pool of human sera collected from individuals living in Papua New Guinea were affinity purified on the immobilized- antigen as described (8) .
  • Protein extracts of cultures of P.falciparum were prepared and fractionated on 7.5% polyacrylamide/NaDodSO. gels. Proteins from the gels were transferred electrophoretically to nitrocellulose, incubated in 5% non-fat milk powder in Pi/NaCl and reacted with affinity purified human antiserum. The filters were incubated with 125I-labelled protein A and autoradiographed.
  • Parasite nuclei were counterstained with propidium iodide and the slides were mounted in 90% glycerol/10% PBS containing p-phenylenediamine for viewing under U.V. illumination.
  • Phage DNA was digested with EcoRI and size-fractionated on a 1% low-melting agarose-gel.
  • the insert(s) wa ⁇ recovered by phenol extraction, subcloned in pUC-9, purified and then nick-translated.
  • Hybridizations were in 0.75M NaCl/0.75M Na citrate/50% formamide/50 ⁇ g ml " salmon sperm DNA/lO ⁇ g ml poly (C)/0.02% Ficoll/0.2% polyvinyl-pyrollidone/0.2% BSA at 42°.
  • the dideoxy chain termination method (9) was employed for sequence determinations.
  • PFG electrophoresi ⁇ (16) wa ⁇ performed e ⁇ entially as described in Kemp et al (11) .
  • Ag57 contains an insert of 1165 bp and exhibits an open reading frame that is in
  • Figure 2 is correct. In addition, all other possible reading frames are interrupted by multiple stop codons.
  • the open reading frame of Ag57 extends from position 2
  • this ATG marks a change in the base composition from 90% AT in the 5' region to 67% in the signal peptide region, a change typical of the start of
  • the deduced amino acid sequence contains 2 blocks of tandemly repeated oligopeptides which encompa ⁇ s most of the coding region.
  • the first block of repeats start ⁇ at po ⁇ ition 203 with 3 tripeptide ⁇ (Ala-Hi ⁇ -Hi ⁇ ) ,
  • a second block of repeats commences. It consists of pentapeptides with the consensus sequence of His-Hi ⁇ -A ⁇ p-Gly-Ala. Repeats 1 and 3 of this block show variation of the 2nd (A ⁇ - G) and 3rd (T ⁇ - A) nucleotide of the fourth codon, replacing Gly by Asp.
  • pentapeptide repeats 1 to 4 Two types of decapeptides encompa ⁇ ing pentapeptide repeats 1 to 4 can be distingui ⁇ hed.
  • the 8th and la ⁇ t pentapeptide repeat of this block is also degenerate and is followed by a termination codon 19 base pairs downstream.
  • the calculated molecular weight for the native .falciparum protein encoded by Ag57 commencing from the AUG is 21,108, which is considerably lower than the apparent molecular weight determined by immunoblotting (see Figure 1) .
  • the actual discrepancy would be even greater if the signal peptide is removed as proposed. We believe that this discrepancy is due to anomalous binding of SDS to the extensive repetitive portions of this molecule.
  • the discrepancy i ⁇ not due to recombination of the repeat ⁇ in E.coli because the distance between the Ahalll sites determined from the sequence of Ag57 is identical to the size of this Ahalll fragment observed by hybridization to genomic DNA (data not shown) .
  • Ag57 codes for a protein which contains 30% histidine and 29% alanine.
  • the library contained cDNA sequences 0.6-2.0kb in length, cloned in the vector ⁇ gtllAmp3( ⁇ mp3) , derived from the Papua New Guinea isolate of P.falciparum FCQ27/PNG (FC27) .
  • FC27 Papua New Guinea isolate of P.falciparum FCQ27/PNG
  • the antibodies that bound to the fixed merozoites were then eluted and reacted with the array of a ⁇ tigen-expres ⁇ ing colonies described above, and also with* randomly-selected colonies.
  • One colony in the NF7 array, designated Ag319 (Fig.4A) reacted much more strongly than any others with these purified antibodies.
  • the human antibodies purified on Ag319 and a rabbit antiserum raised against the purified fused polypeptide from Ag319 were used in immunofluore ⁇ cence a ⁇ says, both on acetone-fixed ⁇ mear ⁇ and on preparation ⁇ lightly fixed with glutaraldehyde and air-dried. Both anti ⁇ era gave identical re ⁇ ults.
  • ARP was detectable by IFAT in acetone-methanol fixed intra-cellular parasites and free parasite ⁇ . It wa ⁇ also detectable on free parasite ⁇ (merozoites) after glutaraldehyde fixation (Fig.6B) . Nucleotide and amino acid sequence of ARP
  • the 1.6 kb cDNA insert from Ag319 and fragments generated from it by digestion with Ahalll or Rsal were subcloned in the vectors Ml3 and mp8 and mp9 and sequenced by the dideoxy chain termination procedure (Fig.7).
  • the sequence of Ag319 shown translated in Figure 8 contains a ⁇ ingle open reading frame that extends throughout the cDNA.
  • the sequence exhibits a very high AT-content with 50% Adenine and 27% Thymidine in the coding strand.
  • ARP contains 40% A ⁇ paragine
  • the insert of Ag319 codes for a relatively hydrophilic polypeptide. Surprisingly, 40% of the
  • Ag319-polypeptide consist ⁇ of a ⁇ paragine and methionine i ⁇ al ⁇ o unusually abundant (7.6%).
  • cDNA clone In the middle of the cDNA clone, commencing at position 803, there are 3 tetrapeptide (Asn-Asn-Asn-Met) and 4 octapeptide repeat ⁇ 18
  • DNA from 5 P.falciparum i ⁇ olate ⁇ (FC27, IMR143, IMR144 and MAD71 from Papua New Guinea and NF7 from Ghana) were cleaved with Ahalll and R ⁇ al, ⁇ ize-fractionated on 1% agarose, blotted to nitrocellulose and hybridized with the Ag319 probe.
  • the result ⁇ (data not ⁇ hown) indicate that ARP doe ⁇ not exhibit re ⁇ triction fragment polymorphi ⁇ m ⁇ like those observed for a number of other cloned P.falciparum antigens (18).
  • the Ag319 probe hybridized to a 0.75 kb Ahalll and to 3.5 and 1.4 kb Rsal-fragments, in accord with the restriction map of Ag319 ( Figure 7) .
  • the block of 3 tetra- and 4 octapeptide repeats is located within a 680 bp Ahalll fragment, which is close to the size of the chromosomal fragment mea ⁇ ured by Southern blotting. This finding is important as it has been shown that approximately 100 repeats were deleted from a chromosomal clone encoding the S-antigen of P.falciparum isolate FC27 (21) .
  • Ag7 encodes an antigen that undergoes processing.
  • Such antibodies were reacted with ummunoblots of lysates from in vitro culture-derived synchronized P.falciparum parasites (Fig.lOA).
  • the major polypeptide detected by these antibodies in mature trophozoite ⁇ and ⁇ chizonts was apparently greater than 250 kilodaltons in size, although there were no accurate markers in this extreme range.
  • Two smaller polypeptides, of approximate sizes 86 and 82Kd, were also detected.
  • the 250Kd band was not present in merozoites but a complex of bands of smaller size with a prominent doublet at 125 and 115Kd wa ⁇ ⁇ een. There wa ⁇ virtually no reactive material detectable in ring ⁇ tage ⁇ .
  • MESA i ⁇ pre ⁇ ent in mature ⁇ tage para ⁇ ite ⁇ a ⁇ a high molecular weight precursor that is specifically processed further to several products in the merozoite and degraded in 20 ring stage ⁇ .
  • Examination of ⁇ upernatant ⁇ from ⁇ ynchronized cultures demonstrated the presence of these processed fragments at the time of schizont rupture (data not shown) .
  • Ag7 does not produce an abundant fused polypeptide detectable by Coomas ⁇ ie blue staining (17) .
  • Clones expressing abundant fused polypeptides were generated by fragmenting and recloning the Ag7 insert.
  • purified cDNA was prepared from the clone Ag7 two inserts were found to be present, as had been found for ⁇ everal other clone ⁇ in thi ⁇ library (17) .
  • the expressing insert wa ⁇ therefore identified by recloning each in ⁇ ert separately into Amp3 and detecting expression in a colony immunoas ⁇ ay using the affinity-purified anti Ag7 antibodies.
  • Ag7 encodes a variable antigen
  • Antibodies prepared against subclone Ag651 were u ⁇ ed to probe immunoblots of lysate ⁇ of a ⁇ ynchronou ⁇ ly grown P.falciparum protein ⁇ (Fig.lOB). Strong reactivity again ⁇ t the 250 kd protein wa ⁇ ⁇ een in . isolates FC27 and NF7 but reactivity against VI and Kl isolates was very much weaker. As well as the differences in intensity, there were also ⁇ light difference ⁇ in ⁇ ize of the ⁇ e large polypeptide ⁇ . When antibodie ⁇ prepared again ⁇ t ⁇ ubclone Ag653 were u ⁇ ed to probe duplicate immunoblot ⁇ a 250Kd band that varied in intensity as above was seen.
  • the nucleotide sequence of Ag7 include ⁇ tandem repeats.
  • the cDNA segment expressing Ag7 was ⁇ elected a ⁇ outlined above, purified and ⁇ ubcloned into M13 vector ⁇ .
  • the ⁇ equence of 681 nucleotide ⁇ contain ⁇ an uninterrupted open reading frame commencing at nucleotide 3 (Fig.11) .
  • Thi ⁇ frame however is out of pha ⁇ e with ⁇ -galactosida ⁇ e, preventing ⁇ ynthesis of a large fused polypeptide.
  • the remaining 124 amino acids flanking the repeat region at the 3' end contain 63 (52%) charged residue ⁇ .
  • __ which encodes a strain specific dominant epitope is composed entirely of the exactly repeated hexapeptide.
  • Genomic DNA of FC27, Kl and NF7 was restricted with either EcoRI or Hindlll, size fractionated by ag ' arose gel electrophore ⁇ i ⁇ and tran ⁇ ferred to nitrocellulose.
  • ag ' arose gel electrophore ⁇ i ⁇ When hybridized with labelled Ag7 cDNA and washed at moderate stringency, a single band was seen in each track (Fig.12) .
  • the size ⁇ of the bands varied considerably, the intensity of hybridization was the same for FC27 and Kl (Fig.12) , as well as VI (data not shown) . This ⁇ ugge ⁇ t ⁇ that there are repeat ⁇ present in all strains.
  • FC27 and VI parasites taken from asynchronou ⁇ in vitro culture were acetone fixed to microscope ⁇ lide ⁇ and examined by indirect immunofluore ⁇ cence.
  • Human anti-Ag651 antibodie ⁇ were used to minimize the effect of cross-reactivity to other proteins.
  • the pattern of reactivity to the intra-cellular para ⁇ ite was similar in both isolates.
  • the localization of Ag7 was also examined using Protein-A immunoeleetronmicroscopy. FC27 parasites derived from asynchronou ⁇ cultures were examined with anti-Ag7 antibodies. Ag7 was detected at the membrane of erythrocytes infected with both trophozoites and schizonts and in membrane lined vesicles -in the red cell cytoplasm (Fig.14) . Ag7 was also detected in some sections associated with the limiting membrane of the intraerythrocytic parasite (data nob shown) . In contrast, the membranes of ring-infected erythrocytes were not labelled, nor were the ring-stage parasites themselves.
  • ring-infected erythrocyte surface antigen (ring-infected erythrocyte surface antigen,. RESA) has previously been described.
  • RESA ring-infected erythrocyte surface antigen
  • a location at the erythrocyte surface similar to that found for RESA but in erythrocytes infected with more mature stages of the parasite leads us to adoption of Mature-parasite-infected Erythrocyte Surface Antigen (MESA) as a name for the present antigen.
  • MSA Mature-parasite-infected Erythrocyte Surface Antigen

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Abstract

Molécules d'ADN comprenant des séquences de polynucléotide construites artificiellement correspondant essentiellement à toute la sequence de base ou à une partie de la séquence de base qui code pour un antigène de Plasmodium falciparum sélectionné dans le groupe formé de l'antigène SHARP, de l'antigène ARP, de l'antigène MESA et des autres antigènes de Plasmodium falciparum produisant une réaction croisée. Lesdites molécules d'ADN peuvent être exprimées comme polypeptide(s). Les peptides ou polypeptides synthétiques présentent l'antigénicité de tous les antigènes SHARP, ARP ou MESA de Plasmodium falciparum ou d'une partie de ceux-ci. Les compositions pour stimuler des réponses immunitaires contre les antigènes de Plasmodium falciparum chez un mammifère comprennent au moins un polypeptide présentant l'antigénicité des antigènes SHARP, ARP ou MESA de Plasmodium falciparum, unis à un support desdites compositions pharmaceutiquement acceptables.
EP19860902288 1985-04-11 1986-04-11 Antigenes hautement repetitifs de plasmodium falciparum. Withdrawn EP0217877A4 (fr)

Applications Claiming Priority (4)

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AU108/85 1985-04-11
AUPH010885 1985-04-11
AUPH164085 1985-07-25
AU1640/85 1985-07-25

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EP0217877A1 true EP0217877A1 (fr) 1987-04-15
EP0217877A4 EP0217877A4 (fr) 1988-03-30

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EP (1) EP0217877A4 (fr)
IL (1) IL78476A0 (fr)
WO (1) WO1986006075A1 (fr)
ZW (1) ZW8386A1 (fr)

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JPS63502002A (ja) * 1985-12-24 1988-08-11 ザ・ワルタ−・アンド・エリザ・ホ−ル・インスティテュ−ト・オヴ・メディカル・リサ−チ 無性の血中段階におけるプラズモジウム・ファルシパルムの抗原
FR2672290B1 (fr) * 1991-02-05 1995-04-21 Pasteur Institut Sequences peptidiques specifiques des stades hepatiques de p. falciparum porteuses d'epitopes capables de stimuler les lymphocytes t.
AU670108B2 (en) * 1992-09-11 1996-07-04 Becton Dickinson & Company Improved antibodies to plasmodium falciparum
ES2894724T3 (es) 2011-12-02 2022-02-15 Rhode Island Hospital Vacuna contra la malaria falciparum

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EP0217877A4 (fr) 1988-03-30
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ZW8386A1 (en) 1986-07-23

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