+

WO1987005607A1 - Polypeptides ayant une immunite protectrice contre la malaria - Google Patents

Polypeptides ayant une immunite protectrice contre la malaria Download PDF

Info

Publication number
WO1987005607A1
WO1987005607A1 PCT/AU1987/000071 AU8700071W WO8705607A1 WO 1987005607 A1 WO1987005607 A1 WO 1987005607A1 AU 8700071 W AU8700071 W AU 8700071W WO 8705607 A1 WO8705607 A1 WO 8705607A1
Authority
WO
WIPO (PCT)
Prior art keywords
resa
sequence
polypeptide
polynucleotide sequence
expression
Prior art date
Application number
PCT/AU1987/000071
Other languages
English (en)
Inventor
Robin Fredric Anders
David James Kemp
Graham Vallancey Brown
Ross Leon Coppel
Graham Frank Mitchell
Graeme Charles Woodrow
Timothy John Tetaz
Neil Howard Goss
Original Assignee
Saramane Pty. Ltd.;
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
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=25643068&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO1987005607(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Saramane Pty. Ltd.; filed Critical Saramane Pty. Ltd.;
Priority to KR1019870701051A priority Critical patent/KR880701243A/ko
Publication of WO1987005607A1 publication Critical patent/WO1987005607A1/fr
Priority to DK598987A priority patent/DK598987A/da

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • 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

  • TECHNICAL FIELD This invention relates to polypeptides which have antigenicity suitable for providing protective immunity against malaria, especially Plasmodium falciparum infections, to processes for the production thereof, vaccines including such polypeptides and immunological methods employing them.
  • RESA Ring-Infected Erythrocyte Surface Antigen
  • the first peptide was 22 amino acids in length and consisted of a tandem repeat of the 11 amino acid sequence DDEHVEEPTVA while the second peptide had the sequence EENVEHDA.
  • the eleven amino acid sequence is part of the so-called 5' repeat region of RESA while the 8 amino acid sequence is found in the 3' repeat region.
  • the present invention is based on the approach that protective immunity to mammalian malaria, especially falciparum malaria,may be induced by immunisation with a vaccine that includes peptides or polypeptides comprising the 5' or 3' repeat region of the RESA antigen of Plasmodium falciparum, or derivatives thereof or parts of those regions or derivatives of parts of those regions or the 11 amino acid and 8 amino acid repeat units or parts or derivatives thereof or combinations or multimers of any of the aforementioned peptides. It is a generalisation that the larger an antigen the more immunogenic it is likely to be. Conversely, small peptides consisting of 30 amino acids or less are unlikely to be immunogenic.
  • small peptides are chemically coupled to larger carrier molecules (e.g. tetanus toxoid, diphtheria toxoid, keyhole limpit haemacyanin) so as to improve their immunogenicity. It is therefore a feasible approach to create a vaccine by coupling 11 amino acid or 8 amino acid peptides (or multiples thereof) that have been chemically synthesised to an appropriate carrier molecule.
  • carrier molecules e.g. tetanus toxoid, diphtheria toxoid, keyhole limpit haemacyanin
  • this invention incorporates not only the synthetic approach to a malaria vaccine but also an approach based on the expression of recombinant molecules, the malaria sequences of which have been derived from the RESA molecule.
  • the present invention provides a polynucleotide sequence which includes: a first polynucleotide sequence which sequence has been derived from the full-length RESA molecule of Plasmodium falciparum, a polynucleotide sequence which hybridizes to said first polynucleotide sequence, a polynucleotide sequence related by mutation, including single or multiple base substitutions, deletions, insertions and inversions to said first sequence or hybridizing sequence or a polynucleotide sequence which on expression codes for a polypeptide derived from the native RESA antigen of Plasmodium falciparum or displays similar biological or imraunological activity to said polypeptide.
  • polynucleotide sequence which is a part, analogue, homologue, derivative or combinations thereof, or multimers of parts, analogues, homologues, derivatives, or combinations thereof of the aforementioned sequence, including the polynucleotide sequences described in the Examples.
  • Also within the scope of this embodiment of the invention is a process for selecting polynucleotide sequences according to the invention which process comprises providing one or more nucleotide sequences and determining which of said sequences hybridizes to a nucleotide sequence known to code for all, part, an analogue, homologue, derivative, multimers or combinations thereof of regions of the RESA antigen of Plasmodium falciparum.
  • the invention provides a probe useful for identification of nucleotide sequences according to the invention which probe comprises a nucleotide sequence derived from regions of the RESA antigen of Plasmodium falciparum or which codes for a polypeptide displaying similar biological or immunological activity to regions of the RESA antigen of Plasmodium falciparum or a sequence which hybridizes to said sequence and a label.
  • a preferred label is a radio-label.
  • the invention provides a process for the production of polynucleotide sequences according to the invention which process may comprise:
  • nucleotide sequences which include the repeat unit of the multimers of polynucleotides according to the invention, and subsequently linking said nucleotide sequences "head to tail" enzymically to form a multimer of repeat units.
  • step (d) inserting said cDNA sequences into an autonomously replicating cloning vector to form a recombinant cloning vector; (e) transforming a host cell with recombinant cloning vector of step (d);
  • step (g) identifying the inserted DNA sequence contained within the cloning vector of said transformed host of step (f);
  • the invention also provides a recombinant DNA molecule characterized by a DNA insert comprising a polynucleotide sequence which is a polynucleotide sequence in accordance with the first embodiment of the invention and vector DNA.
  • the vector DNA of the recombinant DNA molecule comprises plasmid, virus or bacteriophage DNA.
  • Suitable plasmids include pUC13, pSKS106, pLK57, pLV85, pPLc245, ptac12H, pBTA395, pWT571, and pBTA260.
  • Suitable viruses include bovine papilloma virus, adenoviruses, vaccinia retroviruses, baculoviruses, Epstein-Barr virus and SV-40 based viruses.
  • Suitable bacteriophages include M13 and ⁇ . Included within the scope of recombinant DNA molecules according to the invention are recombinant DNA molecules in which an expression control sequence is operatively linked to the DNA. This provides for expression of the molecules in heterologous systems.
  • the invention provides the DNA molecules described in the Examples operatively linked to expression control systems.
  • the invention also provides a transformant host wherein said host is transformed with a recombinant molecule according to the invention.
  • the host cell can be selected from bacterial cells, yeast, fungi and higher eukaryotic cells including plant and human cells.
  • the invention includes transformant hosts capable of expressing polypeptides which are multimers or monomers of all, part, analogues , homologues , derivatives , or combinations thereof of fragments , as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all part, analogues, homologues , derivatives, or combinations thereof of fragments, as well as especially the 5' or 3'repeat units, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum.
  • the invention provides a process for transforming a host so that it carries DNA encoding a polypeptide which is a multimer or monomer of all, part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmouimn falciparum or a polypeptide displaying similar biological or immunological activity to said polypeptide
  • process comprises providing a suitable host and introducing into said host a recombinant DNA molecule according to the invention.
  • an expression product of a transformant host which comprises a polypeptide which is a multimer or a monomer of all, part, analogues, homologues , derivatives , or combinations thereof of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all, part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum.
  • the invention also encompasses such an expression product in a substantially pure form.
  • the expression product can be a fusion product which comprises a first peptide sequence of the transformant host or any other peptide sequence capable of eliciting an increased level of expression in a host or which leads to the production of a more immunogenic molecule and a second peptide sequence which is a multimer or a monomer of all, part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum.
  • the invention also provides a process for the biosynthesis of a polypeptide which polypeptide comprises a multimer or monomer of all, part, analogues, homologues, derivatives, or combinations thereof of various parts, as well as especially the 5' or 3' repeat unit, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all, part, analogues, homologues, derivatives, or combinations thereof of the various parts, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum, which process comprises: providing a recombinant DNA molecule characterized by a DNA insert comprising a first DNA sequence which corresponds to or on expression codes for multimers, or monomers of all, part, analogues, homologues, derivatives, or combinations thereof, of fragments, as well as especially the 5' or 3' repeat units, of the RESA antigen of Plasmodium falciparum or
  • the invention provides a composition for stimulating immune responses in a mammal to the RESA antigen of Plasmodium falciparum, which composition comprises at least one polypeptide comprising a multimer or monomer of all, part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat unit, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar immunological or biological activity to multimers or monomers of all, part, analogues, homologues, derivatives, or combinations thereof of fragments, as well as especially the 5' or 3' repeat unit, of the RESA antigen of Plasmodium falciparum, together with a pharmaceutically acceptable carrier and/or adjuvant or such polypeptide alone.
  • Suitable adjuvants include alhydrogel and various mycobacterial extracts such as muramyl dipeptide.
  • This embodiment also provides a method for manufacturng said composition which method comprises the steps of preparing an effective dosage of at least one polypeptide according to the invention and optionally mixing said effective dosage with a pharmaceutically acceptable carrier and/or adjuvant. Suitable dosage ranges are 0.1 ⁇ g - 3 mg per dose.
  • the invention provides a method of providing immunity to malaria, especially falciparum malaria which method comprises administering to a mammal an effective amount of a polypeptide or composition according to the invention.
  • a further embodiment provides a reagent comprising a polypeptide according to the invention useful in detecting antimalarial antibodies.
  • the invention also provides a method for detecting antimalarial antibodies which method comprises preparing a polypeptide which is a multimer or monomer of all, part, analogues, homologues, derivatives or combinations thereof of regions of the RESA molecule, as well as especially the 5' or 3' repeat unit of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all, part, analogues, homologues, derivatives or combinations thereof of regions of the RESA molecule including the 5' or 3' repeat unit of the RESA antigen of Plasmodium falciparum, and employing said polypeptide in an assay to detect antimalaria antibodies.
  • a further reagent according to the invention comprises antibodies raised against a polypeptide according to the invention, useful in detecting malarial antigens.
  • the invention also provides a method for detecting malarial antigens which process comprises: preparing a polypeptide which is a multimer or monomer of all, part, analogues, homologues, derivatives or combinations thereof of fragments, as well as especially the 5' or 3' repeat unit, of the RESA antigen of Plasmodium falciparum or a polypeptide displaying similar biological or immunological activity to said multimers or monomers of all, part, analogues, homologues, derivatives or combinations thereof, of fragments, especially the 5' or 3' repeat unit of the RESA antigen of Plasmodium falciparum; immunologically challenging an animal with said polypeptide, so as to give rise to antibodies to said polypeptide; and employing said antibodies in an assay to detect malarial antigens.
  • a vaccine composition for immunisation against blood stage P.falciparum antigens in a mammal comprising a synthetic peptide having or including at least one sequence selected from the group consisting of:
  • the synthetic peptide has or includes at least one sequence selected from (DDEHVEEPTVA) n and (EENVEHDA) n wherein n is a positive integer.
  • the present invention is directed to the use of certain peptides, synthesized by Merrifield solid-phase synthesis or other appropriate technology, as repeating oligomers optionally linked to carrier molecules, together with a pharmaceutically acceptable carrier, to stimulate immune responses which protect against the effects of infection with P.falciparum.
  • the vaccine composition of this invention may also include an adjuvant for example, aluminium phosphate, for stimulating the immune response to the synthetic peptide and thereby enhancing the protective effect of the composition.
  • an adjuvant for example, aluminium phosphate
  • the peptides for use in the vaccine compositions of this invention may be prepared by expression in a host cell containing a recombinant DNA molecule which comprises an appropriate nucleotide sequence operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule.
  • the synthetic peptide so expressed may be a fusion polypeptide comprising a portion having or including the desired sequence, and an additional polypeptide coded for by the DNA of the recombinant DNA molecule fused thereto.
  • the synthetic peptides may be produced by chemical means, such as by the well-known Merrifield solid-phase synthesis procedure referred to above (Merrifield, 1963).
  • Figure 1 Detection of RESA-specific protein produced in yeast strain AH22.
  • Strain AH22 containing the expression vector pBTA260 alone (lane A), or containing recombinant vectors where either a 2.8 kb Xmnl fragment of RESA (lane B) or a 3.2kb fragment of RESA (lane C) have been inserted into pBTA260.
  • Lanes D and E show the expression of the 2.8kb Xmnl and the 3.2kb fragments of RESA, respectively, in yeast strain MT302-1c.
  • RESA-specific protein was detected by Western blot analysis, using affinity purified antibodies against the Ag632-Betagalactosidase fusion protein.
  • RESA-specific protein was detected as described for Figure 1, except that the antibody used to detect expression of the 1.3kb EcoRI fragment ( Figure 3B) was a monoclonal against the 3' repeat region of RESA.
  • Figure 3A shows the detection of RESA-specific protein in a Ion- mutant of JM107 containing vector pBTA368 (lane 1) , while the same host strain containing the parent vector pUC13 produces no detectable RESA-specific protein (lane 2) .
  • Figure 3B shows the detection of RESA-specific protein in strain JM109 containing vector ⁇ BTA367 (lane 1), while the same host strain containing vector pBTA395 (a vector closely related to pUC13, differing only in its multiple cloning site) produces no detectable RESA-specific protein
  • FIG. 1 ( lane 2 ) .
  • Figure 4. Detection of beta-galactosidase - Ag632 fusion proteins in E. coli strain JM109. Lanes A and C show the expression of full-length and a shortened derivative of beta-galactosidase (arrowed), produced under control of the expression vectors pSKS106 and pBTA286, respectively. Lanes B and D show the expression of the corresponding beta-galactosidase - Ag632 fusion proteins produced under the control of the expression vectors pBTA224, and a vector closely related to pBTA286, respectively; the latter vector has the multiple cloning site in a different frame to pBTA286 but is otherwise identical to pBTA 286. Fusion proteins were visualized by Coomassie staining of an SDS-polyacrylamide gel containing electrophoretically-separated total cellular proteins.
  • Lane A shows the expression of full-length beta-galactosidase (arrowed) in JM109, produced under control of the expression vector pSKS106, while lane B shows the expression of the beta-galactosidase - Ag28 fusion protein (arrowed) under the control of the expression vector pBTA224.
  • Western blot analysis using a monoclonal antibody against the 3' repeat region, confirms that the band arrowed (in lane B) is RESA-specific (data not shown).
  • FIG. 6 Detection of Ag632B as near-native and as PGK-fusion proteins in yeast strain AH22.
  • Lanes B and D show the expression of Ag632B as a PGK-fusion protein (lane B) in cells containing vector pBTA380, and as a near-native protein (lane D) in cells containing vector pBTA535.
  • Lanes A and C show the lack of RESA-specific protein produced in cells containing the parent vectors pMA27 and pBTA261 (a vector closely related to PBTA260, which differs only in the orientation of the partial EcoRI 2 ⁇ -DNA derived DNA segment), respectively.
  • RESA-specific protein was detected as described in Figure 1, except that a monoclonal antibody against the 5' repeat region was employed.
  • FIG. 1 Western blotting analysis of Ag632B produced under the control of expression vector pLV85 in E. coli strains JM109cI857 (A) and BL459 (B) . The cells were thermo-induced as described in the text. Lanes 1, 3 contain cells grown at
  • Lanes 1 and 2 of (A) and (B) show control strains with the plasmid lacking the insert. The western was probed as described for Figure 7.
  • FIG. 9 Western blotting analysis of native 5' repeats under the control of expression vector pBTA395 in E. coli strains JM101 (A), NB42F' (B), JMl07lon- (C) and JM.109 (D).
  • lanes 1 and 2 show control strain with plasmid lacking the insert; lanes 3 and 4, monomer, lanes 5 and 6, dimer, lanes
  • FIG 10. Western blot analysis of native 5' repeat monomer produced under the control of the expression vector pLV85 in E. coli strain PL459. Lanes A and B show strain PL459 with the plasmid lacking the insert and, lanes C and D, the monomer containing strain grown at 28°C and 42°C respectively. The cells were thermo-induced as described in the text. The western was probed as described for Figure 7.
  • Figure 11. Western blot analysis of synthetic 5' repeats under the control of expression vector pBTA395 in E. coli strains JM101(A), C600 ⁇ (B), TG894(C) and NB42F'(D).
  • lanes 1 and 2 show trimer; lanes 3 and 4, pentamer and lanes 5 and 6, heptamer.
  • the control strain with the plasmid lacking the 5' repeat insert is shown in (C) lanes 7 and 8.
  • (D) lanes 1 and 2 show trimer, lanes 3 and 4 show heptamer.
  • the cells were grown in the absence and presence of IPTG respectively. The western was probed as described in Figure 7.
  • Figure 12 DEAE - Sephacel chromatography of peptide generated by cyanogen bromide cleavage of Ag632
  • the peptides obtained from the cyanogen bromide digestion of the 3-gal-Ag632 fusion product were fractionated on DEAE-Sephacel as described in the text. Peak (C) corresponds to the peptide containing the 5' repeat region.
  • FIG. 14 Predicted structure of the RESA gene and segments that have been expressed in E.coli.
  • the structure shown consists of the sequence of a cloned chromosomal EcoRI fragment from P.falciparum isolate FCQ27/PNG (FC27) (nucleotides 1-3,269) joined at the internal EcoRI site to the sequence of a cloned FC27 cDNA (Ag46 nucleotides 3,270-4,586). The relative positions and end points of fragments expressed in E.coliand used to vaccinate Aotus monkeys are shown.
  • Figure 15 Schematic representation of the 3.59kb fragment of RESA, which is contained in the construct, ⁇ 11.
  • Figure 16 Outline of the pathway for the construction of the yeast expression vectors, pBTA260 and pBTA261.
  • Figure 17 Sequence of the 5' repeat region of RESA that was cleaved from full length RESA DNA using the restriction enzymes HinfI and AluI. The 5' end of the cleaved molecule was converted to the double-stranded form using Klenow enzyme in the presence of the appropriate dNTP's .
  • Figure 18 Sequence of the native 5' repeat region of RESA inserted into the multiple cloning site region of pBTA395 to give plasmid pBTA559. The translated sequence gives the predicted amino acid sequence of the expressed product.
  • Figure 19 Sequence of the synthetic 11 amino acid repeat unit, ligated head-to-tail to form a (33-mer)g DNA molecule to which were added adapters that allowed its ligation to the vector pBTA395. This resulted in vector pBTA540, part of the sequence of which is shown here.
  • the insert termed the monomer, when isolated after digestion with BglII and BamHl formed the basis of furthe head-to-tail self ligations.
  • Figure 20 shows the antibody responses in Aotus monkeys immunized with the fused polypeptide isolated from clone Ag28 (Group 1).
  • Solid lines indicate monkeys that recovered without treatment. Dotted lines indicate monkeys which reached 10% parasitaemia and were treated or died of malaria before reaching 10% parasitaemia.
  • Figure 21 shows the antibody responses in Aotus monkeys immunised with the fused polypeptide isolated from clone Ag632 (Group 2).
  • Figure 22 shows the antibody responses in Aotus monkeys immunised with the fused polypeptides isolated from clones Ag631 and Ag633 (Group 3 ) .
  • BSA antibodies reacting with (EENV) conjugated to BSA.
  • Solid lines indicate monkeys that recovered without treatment. Dotted lines indicate monkeys which reached 10% parasitaemia and were treated or died of malaria before reaching 10% parasitaemia.
  • FIG. 23 Antibody responses measured by micro-ELISA in mice immunized with RESA synthetic peptides.
  • mice were immunized with 100 ⁇ g of KLH conjugate together with complete Freund's adjuvant and boosted four weeks later with the same amount of conjugate in incomplete adjuvant. The mice were bled for antibody measurements two weeks after the boost.
  • the sera were assayed in duplicate at one dilution (1:250) against each of the synthetic peptides conjugated to BSA (upper panel) and fused polypeptides corresponding to the 3' repeat (FPAg28) and 5' repeat (FPAg632) of RESA (lower panel).
  • the results are the averages for five, except for the group immunized with RESA 5'-1Y, where only three mice survived.
  • Figure 24 Reactivity of mouse anti-RESA peptide antisera, measured by micro-ELISA, with a sonicate of erythrocytes infected with
  • E. coli Bacterial cells were grown to early logarithmic phase and induced according to the expression system used. Those containing the lac promoter were grown at 37°C in either a rich
  • Yeast cells were induced by growth to logarithmic phase in minimal medium containing 2% glucose. Cell pellets were collected, and the cells were broken by vortexing with glass beads in distilled water containing ImM phenylmethylsulphonyl fluoride (PMSF) . Total cellular debris was then boiled in sample buffer for 2-5 minutes to denature proteins.
  • PMSF ImM phenylmethylsulphonyl fluoride
  • Samples of total cellular protein from yeast or E. coli were prepared as described above, and separated by one-dimensional electrophoresis through SDS-polyacrylamide gels. Protein bands were then either visualized by staining the gel with Coomassie Blue, or transfered to nitrocellulose sheets for immunoblotting.
  • Proteins were electrophoretically transferred from SDS-polyacrylamide gels to nitrocellulose sheets. The sheets were then incubated in Blotto (5% non-fat milk powder in Phosphate Buffered Saline), followed by incubation with one of the following anti-RESA antibodies:
  • the sheets were subsequently washed, and then either incubated with 125 I-Protein A, washed and autoradiographed; or incubated with anti-rabbit IgG conjugated to alkaline phosphatase, washed and developed with the substrate (5-bromo-4-chloro-3-indolyl-phosphate).
  • Protein A was carried out in Blotto buffered to pH 8.8.
  • the appropriate bacterial cells were grown to mid-logarithmic phase in aerated 800ml cultures, heat-induced at 45°C for 15min and incubated for a further 90min at 37°C.
  • the bacterial cells were collected by centrifugation and lysed by treatment with 0.25mg/ml lysozyme, 10mM Tris, pH8, 2mM EDTA and 50mM NaCl.
  • Triton X-100 was added to a final concentration of 0.2% and the solution was made to 10mM MgCl 2 and lpg/ml DNase. After incubation for 30min at room temperature, cell debris was removed by centrifugation at 850g.
  • Insoluble bacterial proteins were collected by centrifugation at 40,000g, then solubilized in 0.1M phosphate buffer pH 6.8, containing 2% SDS and 10mM dithiothreitol (DTT) .
  • the solubilized proteins were then fractionated by size exclusion chromatography on a Sephacryl S300 column (25mm x 90mm) linked in series with a Sephacryl S400 column (25mm x 90mm) .
  • the columns were equilibrated and eluted with 0.1M phosphate buffer, pH 6.8, containing 0.1% SDS and 1mM DTT at a flow rate of 40ml/hr.
  • the HA was allowed to settle, the supernatant was decanted and discarded, and the HA resuspended in 0.1M phosphate buffer, pH 6.4, containing ImM DTT but no SDS.
  • 0.1M phosphate buffer pH 6.4, containing ImM DTT but no SDS.
  • the fused polypeptides were eluted with 0.5M phosphate buffer, pH 6.8, containing ImM DTT.
  • the fused polypeptides were stored in this solution at -70°C until used.
  • Peptide RESA 5'-1 and RESA 5'-1Y were synthesized by the FMoc solid-phase synthesis methodology of Atherton et al (1983) on a Kieselguhr KA resin support. All other peptides were synthesized using the Merrifield solid-phase TBoc methodology (1963) either manually or on the Applied Biosystems Inc. model 430A automatic peptide synthesizer. The peptides were conjugated to the carrier proteins, keyhold limpet haemocyanin (KLH) and bovine serum albumin (BSA) using glutaraldehyde.
  • KLH keyhold limpet haemocyanin
  • BSA bovine serum albumin
  • mice 0.5ml of 25mM glutaraldehyde was added dropwise to 2mg of peptide and 4mg of protein in 1ml of 0.1M phosphate buffer, pH 7.0, and the solution was then allowed to stand at room temperature for 6 hours. Subsequently, the conjugates were dialysed against several changes of phosphate-buffered saline (PBS) at 4°C for 24hours.
  • PBS phosphate-buffered saline
  • mice were immunized with 100 ⁇ g (carrier protein) of KLH/peptide conjugate together with Freund's complete adjuvant (FCA) and boosted four weeks later with the same amount of conjugate in incomplete adjuvant. The mice were bled two weeks after the boost.
  • FCA Freund's complete adjuvant
  • Rabbits were immunized with 250 ⁇ g (carrier protein) of diphtheria toxoid/peptide conjugate together with FCA, nor-muramyl dipeptide/Squalene-Aracel (Ciba-Geigy) or alum. There were three rabbits in each experimental group and the animals were immunized intramuscularly and three weeks later given a second identical immunization.
  • vaccinia virus plaques were obtained and screened for expression of the 5' end of RESA using a rabbit antiserum specific for the "5' repeats" of the RESA polypeptide.
  • a positive clone, termed ⁇ 11 was sequenced and found to commence immediately after the AUG start codon.
  • Purified DNA of the recombinant vector ⁇ 11 ( Figure 15) was digested with BamHI, and then made blunt-ended by treatment with Klenow enzyme in the presence of dNTPs.
  • a synthetic linker (New England Biolabs, 5' GAAGATCTTC 3") was then ligated to the cut vector which effectively converted the original BamHI site to a BglII site.
  • ⁇ 11 was partially digested with Xmnl and a recombinant plasmid isolated in which the Xmnl site downstream of the RESA coding region was converted to a BamHI site by the use of synthetic linkers.
  • the BglII-Pstl fragment (a. Figure 15 ) and the Pstl-BamHI fragment (b. Figure 15 ) were then isolated by purification from a low melting-point (LMP) agarose gel, and these two fragments were ligated together into the Bglll-site of the yeast expression vector pBTA260, to produce the recombinant vector PBTA369.
  • LMP low melting-point
  • the vectors pBTA230 and pBTA234 were constructed by ligating, into the EcoRl site of pBTA229, a partial EcoRI fragment (containing the yeast 2y plasmid origin of replication and leu-2 gene) from pMA3a (Kingsman, S.M., et al,
  • the recombinant vector pBTA369 containing the 3.2kb full-length RESA fragment, was transformed by the LiCl procedure (Ito, H. , et al, 1983, J. Bact, 153:163-168) into yeast strains AH22 (Bowen, B.A., et al, 1984, Nucleic Acids Research, 12: 1627-1640) and MT302-lc (Mellor, J., et al, 1983, Gene 24:1-14). Isolation of proteins from yeast cultures, and analysis of these proteins by SDS-PAGE and immunoblotting, were done as described in Materials and Methods. Expression of full length RESA in yeast was detectable by Western analysis ( Figure 1, lanes C and E but not by Coomassie staining of SDS-PA gels.
  • a recombinant vector to express full-length RESA in E.coll was constructed via the corresponding yeast vector construct pBTA369 (Example 1(a), above).
  • Purified DNA of pBTA369 was digested with Bglll and BamHI and a 3.9 kb Bglll-BamHI fragment was purified from an LMP agarose gel.
  • This fragment which contained (in addition to the full-length RESA gene): (i) a yeast transcription terminator region from the PGK gene, and (ii) the small Hindlll-BamHI fragment of pBR322, was ligated to the BamHI-digested E. coli expression vector pUC9 (Vieira, J., and J.
  • E.coli strain JM107 Ion- was transformed with both vectors, separately, and the desired recombinant clones were detected by Grunstein hybridisation using as a probe the 1.3kb EcoRI fragment of RESA, which was 32 p-labelled using the random priming method (Feinberg and Vogelstein, 1983, Anal.Biochem., 132:6).
  • Mini-DNA samples front cells bearing plasmid pBTA357 were then prepared and used to transform yeast strains AH22 and MT302-1c. Isolation of proteins from yeast and E. coli cultures, and analysis of proteins by SDS-PAGE and immunoblotting, were done as described in Materials and Methods. Expression of RESA-specific material in both yeast and E. coli was detectable by Western blot analysis ( Figure 1, lanes B and D; Figure 2, lane B). EXAMPLE 3
  • the 1.3 kb EcoRI fragment was purified and ligated to EcoRI-digested pUC13, to produce the recombinant vector pBTA367.
  • the recombinant molecules were used to transform E. coli strains JM107 Ion- and JM109 , respectively.
  • Recombinant clones were detected by Grunstein hybridisation using as probes either the 2.2 kb BamHI-EcoRI or the 1.3kb EcoRI fragment, which were both 32 P-labelled by using the random method.
  • SacI/EcoRI fragment from Ag632 was recloned between the Sad and EcoRI sites of pBTA224 to generate pBTA379.
  • a shorter betagalactosidase fusion to Ag632 was constructed as follows. Purified DNA of pBTA379 was digested with Hpal and Aval, which produced fragments of sizes 4000 bp, 1443 bp, 624 bp and 290 bp. The Aval ends were then made blunt-ended by incubation of the restriction fragments with Klenow enzyme in the presence of dNTPs. The fragments were then separated by electrophoresis through an LMP agarose gel, and the 4 kb fragment was purified and then self-ligated to produce the recom b inant vector pBTA511. This vector is identical to pBTA379 except that a large segment of ⁇ -glactosidase has been deleted.
  • the ligation mix was used as a source of DNA to transform E.coli strain JM109. Isolation of proteins from E.coli cultures and analysis of proteins by SDS-PAGE and immunoblotting were carried out as described in Materials and Methods. Expression of RESA-specific antigenic material was detected, as shown in Figure 4.
  • the vector pBTA286, used as the negative control was derived from vector pBTA224 in the same way that pBTA511 was derived from pBTA379.
  • the chromosomal EcoRI fragment was cloned in ⁇ gt10 and detected with NF7 Agl3L derived from cDNA clone NF7 Ag13
  • NF7 Ag13 (derived from isolate NFS) is colinear with the sequence shown from nucleotide 1,654-3,727; 3 ' to this it has probably undergone deletions in E.coli (Cowman et.al.).
  • the fragments expressed in E.coli (Ag631, Ag632 and Ag633) were generated by sonicating the 5' EcoRI fragment (nucleotides, 1,654-3,269, these correspond to nucleotides 1-1,616 in Cowman et.al.) of clone NF7 Agl3.
  • the fragments were ligated into ⁇ gt11-amp3 after the addition of linkers (GCAATTCC) and screened by hybridization with fragments of NF7 Ag13 to determine which parts of the sequence were present.
  • the probes used were from the EcoRI linker to the Pstl site (nucleotides 1,654-1,969); a 352-base pair (bp) Alul fragment (2,162-2,513) and a 485-bp HincII-EcoRI fragment (2,785-3,269).
  • Clones bearing different portions of the RESA gene were tested for production of stable fused polypeptides by electrophoresis of lysates from the induced clones on SDS-polyacrylamide gels, followed by staining with Coomassie blue.
  • Clones Ag631, Ag632 and Ag633 were selected because they contained a set of overlapping fragments and produced substantial amounts of stable fused polypeptides. The exact end-points of these three fragments were determined by sequencing.
  • Ag631 contains a 989-bp insert (nucleotides 1,654-2,642) and therefore encodes 218 amino acids 5' to the repeats as well as the 5' repeats.
  • Ag632 contains a 997-bp insert (2,208-3,204) and therefore encodes the 5' repeats.
  • the 5' EcoRI site in this clone was destroyed by a cloning artefact; the 5' overhang of the vector EcoRI site was apparently removed and nucleotide 2,208 of the blunt-ended fragment ligated directly to the resulting blunt end.
  • the sequence was therefore determined on a SacI-EcoRI fragment spanning the fusion point using a synthetic oligonucleotide corresponding to the ⁇ -galactosidase sequence.
  • Ag633 contains a 730-bp insert (nucleotides 2,535-3,264) and therefore is located downstream from the 5' repeats.
  • Ag28 has the identical sequence to Ag13 , which has been described in International Patent Specification
  • DNA corresponding to Ag28 was isolated as an EcoRI fragment and ligated into vector pBTA224 that had been cleaved with EcoRI.
  • pBTA224 was derived from pUR292 (Ruther, V. and Muller-Hill, B., 1983, EMBO Journal 2:1791-1794) by removal of the EcoRI site that is located outside the ⁇ -galactosidase gene.
  • the isolated AccI-EcoRI fragment was then briefly digested with Bal31 in order to randomise the ends of the molecule- After the addition of Bglll linkers the molecule (Ag632B) was ligated into the Bglll site of vector pBTA260 (described above) and cloned in E. coli. A11 isolated clones had inserts whose sense strand was in the opposite orientation to that of the PGK translation initiation region.
  • the Ag632B insert from one clone was isolated after digestion with BglII and inserted into the Bglll site of pBTA395.
  • the vector PBTA395 was specially constructed by cloning oligonucleotides between the Sail and BamHI sites in the multiple cloning site of pUC13 to provide the following sites:
  • the resulting recombinant plasmid was termed pBTA.557.
  • the Ag632B insert was then excised from pBTA557 using Sail and BamHI and inserted into the vector pLV85 between the Sail and BamHI sites to yield plasmid pBTA537.
  • the vector pLV85 is identical to the vector pPLc245 (Remaut, E. et al, 1983, Nucleic Acids Research 14.: 4677-4688) except that it has the following multiple cloning site:
  • a fusion of Ag632B to the PGK gene of yeast was constructed as follows. Ag632B was excised from pBTA 557 using Bglll and ligated into the Bglll site found near the 3' end of the PGK structural gene contained on vector pMA27, to yield plasmid pBTA380. After characterisation of the constructs in E.coli, yeast cells of strain AH22 were transformed with the plasmid and the levels of expression of RESA-specific material determined. As shown in Figure 6, lane B, the expression of RESA material is detected. EXAMPLE 9
  • the 5' repeat region of RESA was isolated as a DNA fragment after digestion of RESA with restriction endonuclease Hinfl and Alul.
  • the three base overhang at the 5' end created by the Hinfl enzyme was converted to double strand by "filling in” with dA and dT using Klenow enzyme (see Figure 17).
  • This molecule (after addition of Xhol linkers) was cloned into the Xhol site of an E. coli plasmid (pBTA395) that was specially constructed to contain the following restriction enzyme sites in the polylinker of the plasmid pUC13: Sail - Bglll - Xhol - BamHI
  • This multiple cloning site was also constructed so that on insertion of the HinfI-AluI fragment of RESA into the Xhol site the frame of the ATC in the Bglll and the BamHI sites would be identical. (For sequence of this region of pBTA395 see Example 7.)
  • the resulting recombinant plasmidpBTA559 was then digested with enzymes Bglll and BamHI and the insert isolated. This molecule was then self-ligated in the presence of Bglll and BamHI to give a series of oligomers essentially all aligned as head-to-tail tandem repeats, according to the method described by Willson et al (1985). These molecules were size fractionated and ligated into the Bglll site of vector pBTA395. The recombinant molecules were then transformed into E. coli and screened using oligonucleotide probes. Clones containing an in-frame series of multimers were identified. From among. this series the following molecules were chosen for further study: monomer (i.e.
  • a synthetic DNA molecule whose sequence corresponds to the 11 amino acid repeat of the 5' region of RESA was synthesized. In some cases the third base in a codon was changed to conform to the sequence of the most frequently used codons for highly expressed proteins in yeast and E.coli.
  • This molecule was synthesised as two complementary 33 base oligonucleotides on a solid phase system in an Applied BioSystems DNA Synthesiser. The sequences of the oligonucleotides are as follows:
  • E. coli strains were transformed and the expression of each multimeric species was determined by immunoblotting techniques, as described in Materials and Methods. As shown in Figure 11, RESA-specific material could be detected in most of the strains.
  • EXAMPLE 11 Expression of a multimeric series of synthetic DNA molecules whose unit sequence corresponds to the 8 amino acid repeat sequence of RESA
  • a synthetic DNA molecule whose sequence corresponds to that of one of the repeat units from the 3' region of RESA was synthesised. In some cases the third base in a codon was changed to conform to the sequence of the most frequently used codons for highly expressed proteins in yeast and E.coli.
  • This molecule was synthesised as two complementary 48 base oligonucleotides consisting of two tandem repeats of DNA encoding the 8 amino acid repeat. The sequences of the oligonucleotides together with the amino acids encoded by them are as follows:
  • the mutagenic oligonucleotide was the following: 5 ' -CAG TAA TTT CGT TGA TAT CAG CA-3' .
  • the first target was a Met codon downstream from the 5' repeats (bases 2609-2611 in Figure 14). This codon has been converted to ATC (coding for lie) , creating an EcoRV site.
  • the mutant produces an abundant, stable fusion protein indistinguishable in size or immunoreactivity from Ag632.
  • This work describes the isolation of a fragment of RESA containing the 5' repeat region after isolation of the ⁇ -galactosidase-Ag632 fusion polypeptide and its cleavage with CNBr.
  • the E. coli strain JM109 harbouring the plasmid pBTA379 was grown in 41 of TSB medium containing 0.25mgs/ml of ampicillin for 6h., at which time the A 595 was 1.6. To this culture was then added 10ml of 100mM IPTG to induce synthesis of the ⁇ -galactosidase-Ag632 ( ⁇ -gal-Ag 632) fusion product. The culture was incubated overnight at 37°C on a shaker and the cells then recovered by centrifuging at 17,700 g for 30 min at 4°C. The cell pellet was resuspended in 0.95% saline solution and the cells recovered by centrifuging as described above.
  • the washed cell pellet was resuspended in 0-95% saline solution and passed through a Martin-Gaulin Press three times at 19,000 psi to achieve lysis of the cells.
  • the lysed cell suspension was centrifuged at 17,700 g for 30 min at 4°C, the supernatant discarded and the pellet resuspended in 200ml of 0.1M sodium phosphate, pH7.0, containing ImM EDTA, ImM PMSF and 5% Triton X100. This solution was again centrifuged at 17,700 g for 30 min at 4°C.
  • the ⁇ -ga.l-Ag 632 fusion product which was contained exclusively in the pellet of this centrifugation, was solublized by the addition of 50ml of 0.1M Tris-Cl, pH8.0, containing 8M urea and 0.1M DTT after incubation for 2h at 37°C. The solution was clarified by centrifuging at 25,700 g for 30 rains at 4°C and the supernatant recovered. 3. Sephacryl S-300 chromatography
  • the freeze-dried material was dissolved in 8ml of 70% formic acid and to this added 660mg of cyanogen bromide. The reaction was carried out at ambient temperature in the dark for 24h. and the solution then diluted with 10 volumes of water and freeze-dried.
  • the peptides were then eluted with a linear gradient of sodium chloride from 0 to 400mM in the starting buffer. Upon completion of the gradient the column was eluted with the starting buffer containing 1M sodium chloride (Fig 12). Fractions of 5ml were collected at a flow rate of 3ml/min.
  • Group 3 as in Group 1 there were susceptible and resistant animals which could be discriminated between by their antibody responses to the synthetic peptides.
  • the three resistant monkeys had high responses to the 5' repeat peptide induced by immunizing with the mixture of FP Ag631 and FP Ag633 whereas the two susceptible monkeys (one of which died before reaching the treatment level of 10% parasitaemia) had very much lower antibody levels to this peptide.
  • None of the five animals had antibodies to either 3' repeat peptides induced by immunization with FP Ag631 and FP Ag633, however, consistent with the results in Group 2, the animals were primed for an enhanced response to the (EENV) peptide when the animals were challenged and developed infections.
  • the three control animals that survived to be challenged had no detectable antibodies to any of the three peptides prior to challenge but in two of the three animals there were low levels of antibodies to (EENV) induced by the infections which in two animals rose rapidly to 10% parasitaemia and required treatment and in the other animal rose slowly to approximately 6% parasitaemia which caused death.
  • EENV antibodies to
  • EENV EENVEHDA
  • DDEHVEEPTVAY DDEHVEEPTVAY which is the 5' repeat 11 amino acid sequence from which the other 5' repeat sequences are derived, with a tyrosine added to the C-terminal end (the synthetic peptide used for the Aotus serology was a 22-mer which corresponded to two editions of this 11 amino acid sequence).
  • mice immunized with these peptides were assayed by micro-ELISA against each of the peptides conjugated to BSA, against fused polypeptides corresponding to the 3' and 5' repeats of RESA, and also against sonicates of infected erythrocytes. All mice immunized with these peptides produced antibodies that were reactive with the homologous peptide and the fused polypeptide containing that sequence ( Figure 23) .
  • peptide (EENV) induced antibodies that also reacted with the other 3' repeat peptide, EENVEHDA, which has a five-amino acid sequence in common with it.
  • the peptides used in these experiments were: (EENV) 8 , a 32-mer corresponding to 8 repeats of the RESA 3'-repeat 4-mer; (EENVEHDA) , a 32-mer corresponding to 4 repeats of the RESA 3'-repeat 8-mer; and (DDEHVEEPTVA) 3 , a 33-mer corresponding to 3 repeats of the RESA 5'-repeat 11-mer.
  • Peptides conjugated to diphtheria toxoid with glutaraldehyde were used to immunize rabbits using three different adjuvants: Freund's complete adjuvant (FCA), nor MDP/Squalene-Arlacel (MDP) from Ciba-Geigy and alum. There were three rabbits in each experimental group and the conjugate administered at each immunization was equivalent to 250 ⁇ g of carrier protein. The animals were immunized intramuscularly and three weeks after the primary immunization were given a second booster immunization using the identical dose of adjuvant and route of immunization.
  • FCA Freund's complete adjuvant
  • MDP MDP/Squalene-Arlacel
  • the antibody responses to immunization were determined by solid phase ELISA using as the target antigen the same RESA peptides conjugated to bovine serum albumin.
  • the results in Table 3 are the optical densities obtained with 1:1000 dilutions of the rabbits sera and show that high antibody responses were achieved using FCA and MDP as adjuvants but not with alum as adjuvant.
  • the invention can be used to provide vaccines effective in controlling mammalian malaria, especially falciparum malaria, by providing protective immunity against malaria.
  • the invention can be used for the preparation of reagents useful for detecting malarial antigens, malarial parasites or anti-malaria antibodies.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Des polypeptides, dont l'activité antigénique assure une immunité protectrice contre la malaria, sont obtenus à partir de l'antigène RESA de plasmodium falciparum, et en particulier de la région de répétition (5') et/ou (3'). Des procédés de production desdits polypeptides, des compositions de vaccins comprenant lesdits polypeptides et des procédés immunologiques les utilisant sont également décrits.
PCT/AU1987/000071 1986-03-14 1987-03-13 Polypeptides ayant une immunite protectrice contre la malaria WO1987005607A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
KR1019870701051A KR880701243A (ko) 1986-03-14 1987-03-13 말라리아에 대한 보호 면역을 제공하는 폴리펩티드
DK598987A DK598987A (da) 1986-03-14 1987-11-13 Polypeptider, der tilvejebringer beskyttende immunitet mod malaria

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPH504586 1986-03-14
AUPH5045 1986-03-14
AUPH559586 1986-04-23
AUPH5595 1986-04-23

Publications (1)

Publication Number Publication Date
WO1987005607A1 true WO1987005607A1 (fr) 1987-09-24

Family

ID=25643068

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1987/000071 WO1987005607A1 (fr) 1986-03-14 1987-03-13 Polypeptides ayant une immunite protectrice contre la malaria

Country Status (6)

Country Link
EP (1) EP0297110A4 (fr)
JP (1) JPS63503197A (fr)
KR (1) KR880701243A (fr)
IL (1) IL81890A0 (fr)
WO (1) WO1987005607A1 (fr)
ZW (1) ZW5087A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2199326B (en) * 1986-12-23 1990-11-14 Eniricerche Spa Polypeptidic composition useful for the preparation of antimalarial vaccines and of diagnostic kits for the detection of antimerozoite antibodies
WO1993003057A1 (fr) * 1991-07-31 1993-02-18 Institut Pasteur Polypeptides aptes a induire in vivo des anticorps eux-memes capables d'inhiber l'invasion de globules rouges par des merozoites de p.falciparum, produits apparentes et leur application a la production de compositions vaccinantes
US7438917B2 (en) * 1991-02-05 2008-10-21 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2384284A (en) * 1983-01-28 1984-08-09 Saramane Pty Ltd Expression of plasmodium falciparum polypeptides from cloned cdna
AU4732685A (en) * 1984-09-11 1986-03-20 Saramane Pty Ltd Antigens of plasmodium falciparum
AU5603786A (en) * 1985-04-11 1986-10-16 Walter And Eliza Hall Institute Of Medical Research, The Highly repetitive antigens - sharp-arp-mesa-plasmodium falciparum

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1213576B (it) * 1986-12-23 1989-12-20 Eniricerche Spa Composizione polipeptidica utile per la preparazione di vaccini antimalaria e di kits diagnostici per la determinazione di anticorpi antimerozoite.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2384284A (en) * 1983-01-28 1984-08-09 Saramane Pty Ltd Expression of plasmodium falciparum polypeptides from cloned cdna
AU4732685A (en) * 1984-09-11 1986-03-20 Saramane Pty Ltd Antigens of plasmodium falciparum
AU5603786A (en) * 1985-04-11 1986-10-16 Walter And Eliza Hall Institute Of Medical Research, The Highly repetitive antigens - sharp-arp-mesa-plasmodium falciparum

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF EXPERIMENTAL MEDICINE, Volume 162, issued August 1985, (New York, USA), BROWN G.V. et al., "Localization of the Ring-Infected Erythrocyte Surface Antigen (RESA) of Plasmodium Falciparum in Merozoites and Ring-Infected Erythrocytes". *
MOLECULAR BIOLOGICAL MEDICINE, Volume 2, issued 1984, (ACADEMIC PRESS, LONDON, GB), COWMAN A.F. et al., "The Ring-Infected Erythrocyte Surface Antigen (RESA) Polypeptide of Plasmodium Falciparum Contains Two Separate Blocks of Tandem Repeats Encoding Antigenic Epitopes that are Naturally Immunogenic in Man", pp. 207-221. *
NATURE, Volume 310, issued 30 August 1984, (London, GB), COPPEL R.L. et al., "Immune Sera Recognize on Erythrocytes a Plasmodium Falciparum Antigen Compound of Repeated Amino Acid Sequences", pp. 789-792. *
NATURE, Volume 323, issued 18 September 1986, (London, GB), COLLINS W.E. et al., "Immunization of Aotus Monkeys with Recombinant Proteins of an Erythrocyte Surface Antigen of Plasmodium Falciparum", pp. 259-262. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 83, issued February 1986, (Washington DC, USA), BERZINS K. et al., "Rabbit and Human Antibodies to a Repeated Amino Acid Sequence of a Plasmodium Falciparum Antigen Pf155, React with the Native Protein and Inhibit Merozoite Invasion", pp. 1065-1069. *
See also references of EP0297110A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2199326B (en) * 1986-12-23 1990-11-14 Eniricerche Spa Polypeptidic composition useful for the preparation of antimalarial vaccines and of diagnostic kits for the detection of antimerozoite antibodies
US7438917B2 (en) * 1991-02-05 2008-10-21 Institut Pasteur Peptide sequences specific for the hepatic stages of P. falciparum bearing epitopes capable of stimulating the T lymphocytes
WO1993003057A1 (fr) * 1991-07-31 1993-02-18 Institut Pasteur Polypeptides aptes a induire in vivo des anticorps eux-memes capables d'inhiber l'invasion de globules rouges par des merozoites de p.falciparum, produits apparentes et leur application a la production de compositions vaccinantes

Also Published As

Publication number Publication date
IL81890A0 (en) 1987-10-20
EP0297110A1 (fr) 1989-01-04
ZW5087A1 (en) 1987-07-15
JPS63503197A (ja) 1988-11-24
EP0297110A4 (fr) 1989-12-19
KR880701243A (ko) 1988-07-26

Similar Documents

Publication Publication Date Title
Ståhl et al. A dual expression system for the generation, analysis and purification of antibodies to a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA
US5231168A (en) Malaria antigen
US5792463A (en) Hybrid particle immunogens
EP0175261B1 (fr) Immunogènes à base de particule hybride
Triglia et al. The complete sequence of the gene for the knob‐associated histidine‐rich protein from Plasmodium falciparum.
JP2584733B2 (ja) 原虫類抗原用ｄｎａのクロ−ニング
EP0324647A2 (fr) Vecteur d'expression pour des protéines de fusion et des protéines immunogènes
Cheesman et al. The gene encoding topoisomerase II from Plasmodium falciparum
EP0439056B1 (fr) Vaccin contre la coccidiose
IE62866B1 (en) Recombinant and native group b eimeria tenella immunogens useful as coccidiosis vaccines
EP0307472B1 (fr) Produit immunogene obtenu par expression a partir d'une levure recombinante et procede de purification d'un tel produit.
EP0324648B1 (fr) Vaccins recombinants contre eimeria tenella
US6808711B2 (en) Immunologically active proteins from Borrelia burgdorferi, nucleic acids which encode them, and their use in test kits and as vaccines
IE59791B1 (en) Vaccine against varicella-zoster virus
CA2025230A1 (fr) Antigenes de treponema hyodysenteriae ayant un poids moleculaire de 39 kda et adn encodants
WO1987005607A1 (fr) Polypeptides ayant une immunite protectrice contre la malaria
US5061788A (en) Recombinant malarial polypeptides
Löwenadler et al. A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies
EP0193586B1 (fr) ANTIGENES DE $i(PLASMODIUM FALCIPARUM)
EP0320866A2 (fr) Epitope protecteur immunodominant, compris dans la sous-unité S1 de la toxine de pertussis
STOWERS et al. Immunogenicity of recombinant Plasmodium falciparum rhoptry associated proteins 1 and 2
US5393523A (en) Plasmodium falciparum vaccine comprising a recombinant histidine-rich protein-HRP-II
US5225534A (en) Recombinant malarial polypeptides
AU645482B2 (en) A malaria antigen
PT87137B (pt) Processo de preparacao do produto de expressao de levedura recombinante

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU DK FI JP KR LK MW NO SD US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BJ CF CG CH CM DE FR GA GB IT LU ML MR NL SE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 1987902028

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1987902028

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1987902028

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载