EP0297110A1 - Polypeptides providing protective immunity against malaria - Google Patents

Polypeptides providing protective immunity against malaria

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Publication number
EP0297110A1
EP0297110A1 EP87902028A EP87902028A EP0297110A1 EP 0297110 A1 EP0297110 A1 EP 0297110A1 EP 87902028 A EP87902028 A EP 87902028A EP 87902028 A EP87902028 A EP 87902028A EP 0297110 A1 EP0297110 A1 EP 0297110A1
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EP
European Patent Office
Prior art keywords
resa
sequence
polypeptide
polynucleotide sequence
expression
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.)
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EP87902028A
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German (de)
French (fr)
Other versions
EP0297110A4 (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
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Saramane Pty Ltd
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Saramane Pty Ltd
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Publication of EP0297110A1 publication Critical patent/EP0297110A1/en
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Classifications

    • 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
    • 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
    • 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.

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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.Polypeptides, whose antigenic activity ensures protective immunity against malaria, are obtained from the RESA antigen of plasmodium falciparum, and in particular from the repeat region (5 ') and / or (3'). Methods of producing said polypeptides, vaccine compositions comprising said polypeptides and immunological methods using them are also described.

Description

"POLYPEPTIDES PROVIDING PROTECTIVE IMMUNITY AGAINST MALARIA"
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.
BACKGROUND ART The human malaria parasite Plasmodiuiri falciparum encodes many polypeptides that elicit an immune response in man. Recently, molecular cloning techniques have facilitated the analysis of individual polypeptide antigens that are present in this complex mixture (Kemp et al. 1983). Many CDNA clones expressing these antigens have been isolated by screening with human antibodies Escherichia coli colonies that express the cloned sequences. The production and screening of these clones is described in detail in International Patent Specification WO 84/02917, the disclosure of which is incorporated herein by reference. One particular cloned antigen, termed RESA, for Ring-Infected Erythrocyte Surface Antigen, has been described fully in International Patent Application WO 86/01802, the disclosure of which is incorporated herein by reference. The present invention is based upon the discovery that fragments of RESA as fusion proteins with β -galactosidase can protect monkeys against challenge by a virulent strain of Plasmodium falciparum. Analysis of sera from monkeys involved in this trial demonstrated a strong correlation between protection and antibodies to either of two peptides, the sequences of which were derived from the characteristic repeat sequences of RESA.
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.
Accordingly, 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. As a general procedure 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.
It could be expected that the most immunogenic vaccine will be one which presents a large number of RESA repeat epitopes to the immune system. With peptides that are chemically synthesised there is a practical limit to the length that can be achieved. For a commercial vaccine, a cost-effective peptide would be shorter still, of the order of only 20-30 amino acids. Hence, 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.
DESCRIPTION OF THE INVENTION In a first embodiment, 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.
Within the scope of this embodiment of the invention is included a 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. In accordance with this embodiment, 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. In a second embodiment, the invention provides a process for the production of polynucleotide sequences according to the invention which process may comprise:
1. synthesizing 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.
2. (a) providing a plurality of RNA sequences, one or more of which codes an amino acid sequence which constitutes a peptide or polypeptide unit in accordance with the invention;
(b) synthesizing first strand DNA sequences complementary to said RNA sequences;
(c) synthesizing second strand DNA complementary to said first strand DNA sequences to form double stranded cDNA;
(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);
(f) selecting the transformed host cells in which the DNA sequences inserted encodes a peptide or polypeptide unit in accordance with the invention;
(g) identifying the inserted DNA sequence contained within the cloning vector of said transformed host of step (f);
(h) isolating said inserted cDNA sequence; (i) preparation of appropriate fragments of RESA DNA either by restriction with enzymes, shearing by sonicatlon or digestion with enzymes DNase I or Bal 31 or by combinations of these procedures; and
(j) where appropriate in accordance with the invention enzymatically linking, said fragment "head to tail" to form a multimer of repeating units.
3. Isolating genomic DNA (of Plasmodium falciparum) containing the gene for the RESA antigen and:
(a) preparing various fragments of RESA DNA eitherby restriction with enzymes, shearing by sonication orby digestion with enzymes DNasel or Bal31 or by various combinations of these procedures.
(b) enzymically linking isolated fragments of RESA DNA "head-to-tail" to form inframe multimers of these fragments. It is preferred that the polynucleotide encoding the 8 amino acid repeat region be prepared synthetically as there are no appropriate restriction enzyme sites flanking this region in the native sequence unlike the region encoding the 11 amino acid repeat. Nonetheless this approach does not preclude the isolation of the 8 amino acid repeat region from cloned cDNA or genomic DNA, followed by isolation of a restriction fragment containing the 8 amino acid repeat which is "trimmed" by exonucleases to provide a DNA fragment containing substantially only the 8 amino acid repeat region.
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 λ. 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. In a preferred form 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 which process comprises providing a suitable host and introducing into said host a recombinant DNA molecule according to the invention. There is also provided an expression product of a transformant host according to the invention, 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.
Further 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 a polypeptide which displays 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 units, of the RESA antigen of Plasmodium falciparum; transforming a host with said recombinant DNA molecule so that said host is capable of expressing a proteinaceous product which includes a polypeptide which comprises 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 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; and culturing said host to obtain said expression and collecting the proteinaceous product.
In a third embodiment 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 0.1μg - 3 mg per dose. In a fourth embodiment 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.
According to a different aspect of the present invention, there is provided 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:
(DDEHVEEPTVA) n (EENVEHDA) n and (EENV) n, wherein n is a positive integer, or a related sequence derived therefrom by deletion and/or conservative substitution, said synthetic peptide optionally being coupled to a carrier molecule, together with a pharmaceutically acceptable carrier therefor.
Preferably, the synthetic peptide has or includes at least one sequence selected from (DDEHVEEPTVA) n and (EENVEHDA) n wherein n is a positive integer. As noted above, 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.
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. Alternatively, 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).
BRIEF DESCRIPTION OF THE DRAWINGS
Further details of the present invention will be apparent from the detailed description hereunder, and from the accompanying Figures: 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.
Figure 2. Detection of RESA-specific protein expressed in a Ion- muta derivative of E. coli strain JM107 containing vectors pSKS106 (lane A), pBTA354 (lane B), pUC9 (lane C) or pBTA356 (lane D). RESA-specific protein was detected as described for Figure 1.
Figure 3. Detection of RESA-specific protein expressed in E.coli.
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 ρBTA367 (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
( 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.
Western blot analysis, using a monoclonal antibody against the 5' repeat region, confirms that the fusion proteins
(arrowed lanes B and D) are RESA-specific (data not shown).
Figure 5. Detection of a beta-galactosidase-Ag28 fusion protein in
E. coli strain JM109. 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).
Figure 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 2μ-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. Figure 7. Western blotting analysis of Ag632B under the control of expression vector pBTA395 in E. coli strains JM101 (lanes C, D); JM109 (lanes E, F); JM107 lon- (lanes G, H) ; NB42F' (lanes I, J); NB42 (lanes K, L); TG894 (lanes M, N), and AKEC28 (lanes O, P) grown in the presence and absence of IPTG respectively. Lanes- A and B show E. coli strain NB42 with the plasmid lacking the insert. The western was probed with an affinity purified antibody against a β-galactosidase fusion of Ag632 and the bound antibodies detected with sheep anti-rabbit IgG-alkaline phosphatase conjugate.
Figure 8. 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
28°C and lanes 2, 4 cells grown at 42°C. 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.
Figure 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). (A) and (D), lanes 1 and 2 show control strain with plasmid lacking the insert; lanes 3 and 4, monomer, lanes 5 and 6, dimer, lanes
7, 8 trimer and lanes 9, 10, hexamer. (B) and (C); lanes 1, 2, monomer, lanes 3, 4, dimer, lanes 5, 6 trimer and lanes 7,
8, hexamer. The cells were grown in the absence and presence of IPTG respectively. The western was probed as described for Figure 7.
Figure 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), C600λ(B), TG894(C) and NB42F'(D). (A), (B) and (C); 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.
Figure 13 Western blot analysis of peak (C) from the DEAE-Sephacel chromotraphy
An aliquot of the fraction containing peak C from Figure 12 was electrophoresed on a 20% SDS-PAGE and then transferred to nitrocellulose as described in the text. The band was detected using a monoclonal antibody (Mab 306.14) raised against the fused protein produced by clone Ag632 (FP Ag632) and a anti-mouse IgG antibody coupled to alkaline phosphatase.
Figure 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, Δ11.
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).
A. antibodies reacting with EENVEHDA conjugated to BSA.
B. antibodies reacting with (EENV)4 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. Figure 21. shows the antibody responses in Aotus monkeys immunised with the fused polypeptide isolated from clone Ag632 (Group 2).
A. antibodies reacting with (DDEHVEEPTVA) conjugated to BSA.
B. antibodies reacting with (EENV) conjugated to BSA.
All monkeys recovered without treatment.
Figure 22. showsthe antibody responses in Aotus monkeys immunised with the fused polypeptides isolated from clones Ag631 and Ag633 (Group 3 ) .
A. antibodies reacting with (DDEHVEEPTVA) conjugated to
BSA. B. 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.
Figure 23 Antibody responses measured by micro-ELISA in mice immunized with RESA synthetic peptides. The peptides, coupled to KLH, were: EENVEHDA (RESA 3'-1), solid bars; (EENV)4 (RESA
3'-2), hatched bars; DDEHVEEPTVAY. (RESA 5'-1-f), shaded bars, and as a control NF7S-1, a 16-amino acid peptide corresponding to two repeats of the P.falciparum isolate NF7 S antigen, open bars. Mice were immunized with 100μg 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
P.falciparum (isolate FC27). Each bar is the value obtained for an individual mouse.
Deposits of strains designated herein as pBTA382, pBTA535, pBTA379, pBTA537 and pBTA356 have been made at the American Type Culture Collection, Bethesda, Maryland, U.S.A, on under accession numbers Materials and Methods
Preparation of total cellular protein extracts
(a) 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
(tryptone soya broth) or minimal (Mq salts plus glycerol) medium, induced with ImM isopropyl-thio-galactoside (IPTG) and allowed to grow a further 2-4 hours before harvesting. Cells containing the λPL promoter (with the temperature-sensitive repressor cI857) were initially grown in rich medium at 30°C, and then induced by transfer to 42°C for 4 hours. Cells were then centrifuged, and the pellets were resuspended in electrophoresis sample buffer and boiled for 5-15 minutes to denature proteins.
(b) Yeast : 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.
Analysis of total cellular protein by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
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.
Analysis of proteins by 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:
(i) rabbit antiserum against a beta-galactosidase-Ag632 fusion protein (preadsorbed against E. coli and yeast extracts). (ii) affinity-purified antibodies against beta-galactosidase-Ag632 fusion protein, or (iii) monoclonal antibodies against either the 5' or 3' repeat region.
The sheets were subsequently washed, and then either incubated with 125I-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).
Since the monoclonal antibody to the 5' repeat region was unable to bind Protein A, an intermediate incubation with affinity-isolated sheep anti-mouse IgG (Silenus) enabled the use of 125I-Protein A in immunoassays with this monoclonal. In such cases, incubation with
Protein A was carried out in Blotto buffered to pH 8.8.
DNA methods
All methods used in the isolation and manipulation of DNA were essentially as described in Maniatis et.al., 1982, "Molecular Cloning", Cold Spring Harbor Laboratory. Bacterial strains used.
The following E.coli strains were used: Designation Reference Genotype
JM101 Δ(lac,pro), supE(GlnV), thi/F' proA+B+, lacIq, Δ(lacZ)M15, lacY+A+ , traDl
JM107 2 JM101 hsdRk17, endAl , gyrA96 , relAl
JM109 2 JM107 recA
NB42 3 ED8654 zaj : : Tn5 lonl
NB42F' 1,3 NB42/F' pro A+B+, IacIq, Δ(lacZ)M15, lacY+A+, traDl
TG894 thiA1 , thr-1 , leuB6 , proA2, aryE3 , his-4, lacY1, galK2 , ara-14, xyl-5 , mtl-1, rpsL31, tsx-33, lon-1 (non-mucoid), thyA
AKEC28 thrC, leuB6 , thyA, trpC1117, hsdRk, hsdMk C600(λ) thiA1, thr-1 , leuB6 , supE (glnV)44, lacY1, FhuA21(λ)+
Refs: 1. Gronenborn, B., and Messing, J. (1978) Nature 272:375-377.
2. Yanisch-Perron, C. , Viera, J. , Messing, J. (1985) Gene 33: 103-119.
3. Murray, N.K. et.al. (1977) Molec.Gen.Genet. 150:53-61.
4. Dobson, et.al. (1982) Nucl.Acids Res. 10:2625-2637.
5. Appleyard, R.K., (1954) Genetics 39:440-452. Isolation of fused polypeptides
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 MgCl2 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. Small aliquots of each fraction were electrophoresed on SDS-polyacrylamide gels to identify the elution position of the fused polypeptides. Those fractions containing fused polypeptide reasonably free of other polypeptides were pooled. The fused polypeptides were concentrated from this solution and freed of approximately 90% of contaminating SDS by adsorption onto hydroxyapatite (HA) . For this the pooled fractions were mixed for 20 min at room temperature with HA which had been washed in 0.01M phosphate buffer, pH 6.4, containing 0.1% SDS and ImM DTT. Subsequently 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. After the HA was packed into a 200mm x 15mm column 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.
Synthesis and conjugation of peptides
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. For this, 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. Immunization of mice
Mice were immunized with 100μg (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.
Immunization of rabbits
Rabbits were immunized with 250μg (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.
Antibody measurements using enzyme-linked immunoabsorbent assays (ELISA) .
Conventional micro-ELISAs were performed using flexible polyvinyl chloride microtitre plates coated overnight at 4°C with 50ul of the BSA/peptide conjugates (5μg/ml of BSA) in PBS. Sera were assayed in duplicate at one dilution (1:250 for mice and 1:1000 for rabbits) and the bound antibodies were detected with affinity-purified (on mouse or rabbit immunoglobulin) sheep F(ab) conjugated to horseradish peroxidase. Monkey sera were assayed in triplicate at one dilution (1:2000) and the bound antibodies were detected with affinity-purified (on Aotus immunoglobulin) rabbit F(ab) conjugated to horseradish peroxidase. We employed 2,2'-azinobis (3-ethylbenz-thiazoline sulfonic acid) as the substrate for detecting bound horseradish peroxidase.
Vaccine trial in Aotus monkeys
Animals used in the trial were pre-conditioned in the animal facility of the Malaria Branch, Centers for Disease Control, Atlanta, USA, for a minimum of one month prior to the commencement of the trial which was carried out in the same facility. During this period baseline serologic, haematologic, blood chemistry, and clinical parameters were established. Assignment of animals (Aotus monkeys of karyotypes V, X or XI) to vaccine or control treatment groups was by random allocation utilizing a table of random numbers. Each animal was given two inoculations, 6 weeks apart, of a test or control antigen. For the first immunization, 250μg of the antigen was emulsified in complete Freund's adjuvant and injected intramuscularly. For the second immunization, the same amount of antigen was emulsified in incomplete Freund's adjuvant supplemented with complete Freund's adjuvant, 1 part in 10, and also injected intramuscularly.
Two weeks after the second immunization the animals were challenged by intravenous inoculation of 500,000 asexual parasites obtained from an Aotus monkey infected with the Indochina I/CDC strain of P.falciparum. From the day following challenge, daily blood smears were made for determination of parasitaemia. Serum samples, for estimation of antibody levels, were obtained each week throughout the trial. Animals that developed parasitaemias of 10% or greater were withdrawn from the study and treated with mefloquine.
RESULTS
EXAMPLE 1
Construction of Recombinant Vectors for Expression of full-length
RESA in Yeast and E. coli.
(a) Yeast
In order to facilitate the subcloning of a full-length RESA fragment into both yeast and E. coli expression vectors, an initial construct termed Δ11 was made.
Varying amounts of the 5'-extragenic sequence from the RESA chromosomal EcoRI fragment (Figure 14) were removed with the exonuclease Bal31, and BamHI linkers were added to the new 5' ends. Ten Bal 31 deletion clones were obtained and their deletion end points determined by sequence analysis. Each of the ten BamHI-EcoRI fragments were transferred into pGS62, the plasmid used for vaccinia cloning. Two pools were prepared for transfection into vaccinia infected cells: pool 1 contained the five longest BamHI-EcoRI fragment sub-clones, pool 2, the five shortest. After transfection, 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 Δ11, was sequenced and found to commence immediately after the AUG start codon. Purified DNA of the recombinant vector Δ11 (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. Separately, Δ11 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. Figure15) and the Pstl-BamHI fragment (b. Figure15) 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.
A description of the construction of vectors pBTA260 and pBTA261 follows (see also Figure 16). A 3 kb HinuIII fragment, containing the entire yeast phosphoglycerate kinase (PGK) gene together with the PGK transcription and termination control regions, was excised from pMA27 (Mellor, J., et al, 1983, Gene 24: 1-14) and ligated to Hindlll-digested pBR322 (Bolivar, F., et al, 1977, Gene 2: 95-113), to produce the recombinant vector pBTA212. A fragment (approx. 1.2kb in size) was then deleted from this vector, which extended from the Sau3A site immediately upstream from the PGK translation initiation codon through to the unique
Bglll site within the PGK gene, to produce the vector pBTA229. 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,
1983, Gene Expression in Yeast. Proceedings of the Alko Symposium
Helsinki 1983, ed by M. Korhola and E. Vaisanen. Foundation for Biotechnical and Industrial Fermentation Research 1:95-114) . The resultin vectors are identical except for the orientation of this fragment. Finally, a 56 bp double-stranded oligonucleotide was ligated into the unique BglII site of pBTA230 and pBTA234 to produce the PGK promoter-based expression vectors pBTA260 and pBTA261. The nucleotide sequence around the PGK translation initiation region in pBTA260 and pBTA261 is shown below: The DNA sequence upstream of the -10 position is identical to the native sequences.
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.
(b) E.Coli
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. Messing, 1982, Gene 19: 259-268), and recombinant vectors were detected (using the blue/white Xgal procedure) in the transformed E. coli host JM107 Ion (Yanisch-Perron, C, et. al, 1985, Gene 33: 103-119). The recombinant vector is known as pBTA356. Isolation of proteins from E. coll 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 E. coli was detectable by Western blot analysis (Figure 2), but not by Coomassie staining of SDS-PA gels.
EXAMPLE 2
Construction of Recombinant Vectors for Expression of the 2.8kb Xmnl fragment of RESA in Yeast and E. coli.
Purified DNA of Δ11 was digested with Xmnl, and the 2.8kb fragment containing both the 5' and 3' repeat regions of RESA was purified from an LMP agarose gel. Synthetic linkers (New England Biolabs, Bglll, 5' CAGATCTG 3') were then employed to clone this fragment into either the Bglll site of the yeast expression vector pBTA260, or into the BamHI site of the E. coli lac expression vector pSKS106 (Shapira, S.K., et. al., 1983, Gene 25 71-82). The two recombinant vectors thus created are known as pBTA357 and pBTA354, respectively. 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 32p-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
Construction of Recombinant Vectors for the Expression of the 2.2kb BamHI-EcoRI and 1.3kb EcoRI fragments of RESA in E. coli. Purified DNA of vector Δ11 was digested with BamHI and EcoRI, and the fragments were separated by electrophoresis through an LMP agarose gel. The 2.2kb BamHI-EcoRI fragment (which contains the 5' repeat region of RESA, but not the 3' repeat region) was purified and ligated to BamHI-EcoRI double-digested pUC13 (Vieira, J. , and J. Messing, 1982, Gene 19: 259-268), to produce the recombinant vector pBTA368. 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 32P-labelled by using the random method.
Isolation of proteins from E. coli cultures, and analysis of proteins by SDS-PAGE and immunoblotting, were done as described in
Materials and Methods. Expresssion of RESA-specific material in E. coli was detectable by Western blot analysis (Figure 3A, lane 1 and Figure 3B, lane 1).
EXAMPLE 4
Construction of Recombinant Vectors for the Expression of β-galactosidase- Ag632 fusion proteins in E.coli.
The 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 recombinant vector pBTA511. This vector is identical to pBTA379 except that a large segment of β-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.
EXAMPLE 5
Construction of Recombinant Vectors for the Expression of the
RESA Fragments Ag631, Ag632 and Ag633 in E.coli.
The chromosomal EcoRI fragment was cloned in λ gt10 and detected with NF7 Agl3L derived from cDNA clone NF7 Ag13
(overlapping the internal EcoRI site (see Cowman et.al.). All clones were sequenced in full by the chain-termination procedure (Sanger et.al. ) . 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 λ 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 β-galactosidase sequence. Ag633 contains a 730-bp insert (nucleotides 2,535-3,264) and therefore is located downstream from the 5' repeats.
EXAMPLE 6
Construction of Recombinant Vectors for the Expression of the RESA 3' Fragment Ag28 in E.coli.
Ag28 has the identical sequence to Ag13 , which has been described in International Patent Specification
Number WO 84/02917. 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 β-galactosidase gene.
This was achieved by partial digestion of pBTA224 with EcoRI followed by the filling in of the cohesive ends with Klenow enzyme in the presence of the four dNTP's, re-ligation and isolation of a plasmid that had lost the EcoRI site found outside the lac 2 gene.) The ligation mix was used as a source of DNA for the transformation of E.coli strain JM109. Appropriate clones were detected in Grunstein hybridisation experiments using 32P-lab,elled RESA DNA.
Expression of Ag28 as a fusion to β-galactosidase was then checked using procedures described in Materials and Methods. Figure 5, lane B shows the detection of this RESA fragment on a Coomassie stained protein gel. The identification of these molecules was determined using immunoblotting techniques (data not shown) .
EXAMPLE 7
Expression of Ag632B as a Near Native Molecule in E. coli and Yeast
Experiments aimed at the expression of RESA fragments that are not fused to near full length β-galactosidase have been carried out. The prototyp of such molecules is a fragment that approximates in sequence to Ag632. This molecule, termed Ag632B, was derived from RESA as follows. Cloned RESA genomic DNA was cleaved with AccI and EcoRI, as these two sites map near the endpoints of Ag632. 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:
Sail Bglll Xhoi BamHI AGG TCG ACT CAG ATC TAC CTC GAG TTG GAT CCC DNA sequence outside this region is identical to that of pUC13.] 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:
ATG TCG ACG GAT CCA TCG ATA TCG TTA ACA AGC TT...
Sail EcoRV HindIII
BamHI Hpal
Clal
Finally, the Ag632B insert in pBTA557 was excised using Bglll and ligated into the Bglll site of pBTA260 to yield pBTA535.
The recombinant plasmids pBTA557, pBTA537 and pBTA535 were then transformed into the appropriate E.coli and yeast strains, as described in the legends to Figures 6, 7 and 8, and the levels of expression of Ag632B were determined as described in Materials and Methods. As shown in these figures, RESA-specific material could be detected in each case (Figure 6, lane D; Figure 7, lanes C, E, G, I, K, M and 0; Figure 8A, lane 4; Figure 8B, lane 4). EXAMPLE 8 Expression of Ag632B as a fusion to Yeast Phosphoglycerate Kinase
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
Expression of the native 5' repeat region in near native form and as a series of head-to-tail multimers
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. pBTA559, Figure 18), dimer, trimer and hexamer. Some of these multimeric inserts were then cloned into the vector pLV85 as follows. The pBTA395 series of multimer-containing plasmids were digested with Sail and BamHI, and the inserts ligated into pLV85 that had been cut with Sail and BamHI. This not only results in a series of plasmids with the RESA repeat region under the control of the PL promoter but also provides a TAA stop codon immediately downstream of the inserts.
The appropriate E. coli strains were transformed and the expression of each multimeric species was determined by immunoblotting techniques, as described in Materials and Methods. Results in Figure 10 for the monomer show the detection of RESA specific material.
EXAMPLE 10
Expression of a multimeric series of synthetic DNA molecules whose unit seguence corresponds to the 11 amino acid repeat sequence of RESA.
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:
Asp Asp Glu His Val Glu Glu Pro Thr Val Ala
5' - GAT GAC GAA CAC GTT GAA GAA CCA ACT GTT GCT - 3'
3'- CTT GTG CAA CTT CTT GGT TGA CAA CGA CTA CTG - 5'
After annealing to give a double stranded molecule, the DNA sample was mixed with ligase which resulted in a range of long multimeric molecules. The following adaptor molecules were then added to make them compatible for cloning into pBTA395 digested with Bglll and BamHI: a) Adaptor for 5' end
5' - GA T CTA C G T T G C T - 3'
3' - A T G CA A CGA C TA CTG - 5'
b) Adaptor for 3' end
5' - G A T G A C GA TA T C T G - 3'
3' - C TA TA G A C C TA G - 5'
One recombinant molecule was isolated that on DNA sequencing was shown to contain six head to tail units of the basic 33-mer sequence, and was termed pBTA . This plasmid was then digested with Bglll and BamHI and the insert isolated. This molecule was then self-ligated in the presence of BglII and BamHI using the method of Willson et.al. to give a series of oligomers all essentially aligned as head-to-tail tandem repeats. Using methods described in Example 9, a series of in-frame head-to-tail multimers were identified. From amongst this series the following molecules were chosen for further study: trimer, pentamer and heptamer.
Some of these multimeric inserts were then cloned into the vector pLV85, using the same procedure as described in Example 9.
The appropriate 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:
Glu Glu Asn Val Glu His Asp Ala Glu Glu Asn Val Glu His Asp Ala
5' - GAA GAA AAC GTT GAA CAC GAC GCT GAA GAA AAC GTT GAA CAC GAC GCT - 3'
3' - TTG CAA CTT GTG CTG CGA CTT CTT TTG CAA CTT GTG CTG CGA CTT CTT -5'
Synthesis was carried out in an Applied BioSystems DNA Synthesiser. After annealing to give a double-stranded unit, the DNA sample was mixed with ligase which results in a range of long multimeric molecules.
The following adaptor molecules were then added to make them compatible for cloning into pBTA395 that had been digested with Bglll and BamHI.
a) Adaptor for 5' end
5' - G A T C TA C G T T G C T - 3'
3' - A TG CA A C G A C T T C T T - 5'
b) Adaptor for 3' end
5' - G A A G AA G A TA T C T G - 3'
3' - C T A TA G A C C TAG - 5'
One recombinant molecule was isolated that on DNA sequencing was shown to contain three head-to-tail units of the basic 48-mer sequence, and was termed pBTA551. Starting with the insert of this molecule a series of oligomers all aligned as head-to-tail tandem repeats of the starting molecule were constructed and characterised as described in Example 10. From amongst this series the following molecules were chosen for further study: pentamer and decamer.
Some of these multimeric inserts were then cloned into the vector pLV85, using the same procedure as described in Example 9.
The appropriate E. coli strains were transformed. Expression can be determined in the usual way. EXAMPLE 12
Construction and expression of variants of Ag632 in which certain methionine residues have been mutated to alternative amino acids using in vitro mutagenesis techniques A strategy is outlined below for the production of protein fragments of RESA by cyanogen bromide cleavage of the relatively stable Ag632 (a β-galactosidase fusion protein bearing the 5' repeats and flanking sequences). As cleavage of native Ag632 would yield a 5' repeat-bearing fragment of ~110aa, a protein substantially smaller than the ~330aa present in Ag632, we decided to remove selected Met residues by site-directed in vitro mutagenesis. This was performed essentially by the method of Zoller and Smith (1983) on a single-stranded M13 template (mpl9) containing the Sac-EcoRI fragment from clone Ag632. 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.
Similar strategies are applicable for removal of the other Met residues in Ag632. EXAMPLE 13
Isolation of fragments of RESA expressed as part of a larger molecule fused to beta-galactosidase and cleaved therefrom using CNBr.
This work describes the isolation of a fragment of RESA containing the 5' repeat region after isolation of the β-galactosidase-Ag632 fusion polypeptide and its cleavage with CNBr.
1. Growth of cells The E. coli strain JM109 harbouring the plasmid pBTA379 (see Example 4) was grown in 41 of TSB medium containing 0.25mgs/ml of ampicillin for 6h., at which time the A595 was 1.6. To this culture was then added 10ml of 100mM IPTG to induce synthesis of the β-galactosidase-Ag632 (β-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.
2. Lysis and recovery of the β-gal Ag 632 fusion product 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 β-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
To 40ml of the clarified supernatant was added 2.0ml of glacial acetic acid to lower the pH to ca . 4. This solution was then applied to a Sephacryl S-300 column (3.4cm x 80 cm; Vt = 726ml) equilibrated in 0.1M sodium acetate, pH 5.0 containing lmM DTT and 8M Urea and eluted with the same buffer. Fractions of 5.0ml were collected at a flow rate of 1.0 ml/min. The β-gal Ag 632 fusion product eluted at the void volume of the column, as determined by SDS-PAGE, and was dialysed against water and freeze-dried.
4. Cyanogen Bromide digestion of β-gal-Ag 632 fusion product
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.
5. DEAE-Sephacel chromatography
The freeze-dried cleavage products (41.4mg) were dissolved in 20ml of 50mM Tris-Cl, pH7.5, containing 8M Urea, ImM DTT and ImM EDTA and applied to a DEAE-Sephacel column (4.4cm x 12.0cm; Vt = 182 ml) equilibrated in the same buffer. 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. The fractions were examined by Western blot analysis and revealed that the major immunologically cross reactive band was eluted with the 1M sodium chloride wash (Fig.13 ). This is consistent with the known amino acid composition of the cyanogen bromide peptide containing the 5' repeat region. EXAMPLE 14
Testing of the protective ability of various fragments of RESA when monkeys, previously vaccinated with this RESA material, are challenged with a virulent isolate of Plasmodium falciparum. A vaccine trial to test various regions of the RESA molecule has been carried out in collaboration with personnel in the Malaria Section of the Centers for Disease Control in Atlanta, USA. A total of 20 monkeys were used in this trial: 3 test groups (5 monkeys/group) received fused polypeptides produced by the four cDNA clones indicated in Figure 14, and a control group which was immunized with an irrelevant fused polypeptide (see Table 1) .
TABLE 1
Group Immunogen Source of Immunogen
1 FP Ag28 3' repeat of RESA (Isolate FC27)
2 FP Ag632 5' repeat of RESA (Isolate NF7)
3 FP Ag631+ Non-repeat sequences on either
FP Ag633 side of the 5' repeat of RESA + the 5' repeat (Isolate NF7) .
4 FP TA10 A fused protein containing uncharacterized sequences of a Taeniid protein.
Animals were immunized on Day 0 with 250μg of the various antigens together with complete Freund's adjuvant. After 6 weeks the animals were given a second immunization with the same amount of antigen together with incomplete Freund's adjuvant containing 1 part in 10 of complete Freund's adjuvant. Two weeks after this boosting immunization the animals were challenged with 0.5 x 106 Aotus erythrocytes infected with the P.falciparum isolate Indochina 1. The course of infection was monitored daily by microscopic examination of Giemsa-stained blood smears. Animals in which 10% or more of the red blood cells became infected were immediately withdrawn from the trial and treated with mefloquine. The principal measure of efficacy afforded by immunization was the experimental group mean maximum parasitaemia compared to that occurring in the control group. Unfortunately two of the monkeys in the control group (Group 4) and one monkey in Group 2 died before being challenged with P.falciparum, nevertheless a significant effect of immunization was observed (Table 2).
TABLE 2
Peak Difference from
Group Group Mean
Parasitaemias Control
1 10.2*
1.9
0.4 8.4% p > 0.05
16.0* 14.3*
2. 1.8
3.7 2.3% p < 0.05
1.6
2.2
3. 10.2*
2.8
0.08 4.8% p > 0.05
7.5#
3.6
4. 5.9#
10.5* 10.1%
13.0*
* Animals requiring chemotherapy.
# Animals dying of malaria before reaching 10% parasitaemia. Serological Response to Immunization and Challenge Infection.
The significance of this encouraging outcome was dramatically emphasized by the results obtained when the monkeys were examined for antibody responses to different parts of the RESA polypeptide. Serum antibodies were measured by ELISA using synthetic peptides corresponding to 3 different epitopes in RESA and conjugated to bovine serum albumin as the target antigens in solid-phase ELISAs. Two of the peptides, EENVEHDA and (EENV)4 corresponded to the two major repetitive sequences found within the 3' repeat structure of RESA. The other peptide, DDEHVEEPTVA corresponds to a repetitive sequence found in the RESA 5 ' repeat structure. This sequence repeats once but the other repeats are derived from this sequence by deletions and/or conservative substitutions.
The results of these antibody measurements are shown in Figures 20-22. All five monkeys which were immunized with FP Ag28 had high levels of antibodies to the (EENV)4 peptide regardless of whether or not they were rendered resistant (recovered without reaching 10% parasitaemia) or were still susceptible (died of malaria or reached 10% parasitaemia and given mefloquine). Antibodies to (EENV) rose quickly after the primary immunization and were maintained at a high level throughout the course of the trial. When the bleeds from these monkeys were assayed on the other 3' repeat peptide (EENVEHDA) the two animals that were rendered resistant by immunization with FP Ag28 were found to have high levels of antibodies whereas the three susceptible monkeys had undetectable or very low levels of antibodies. No antibodies to the 5' repeat peptide were detected in the sera of these monkeys either before or after they developed patent infections as a result of challenge.
In the four monkeys in Group 2 , all of which were rendered resistant by immunization with FP Ag632, there was a rapid rise in antibodies to the 5 ' repeat peptide (DDEHVEEPTVA) induced by the primary immunization. None of the monkeys in Group 2 developed detectable levels of antibodies to either of the 3' repeat peptides as a result of immunization with Ag632, however, the onset of parasitaemia was associated with the production of antibodies to (EENV)4. Immunization with the 5' repeat fused polypeptide (FP Ag632) clearly primed the animals for an enhanced antibody response to (EENV)4 because the level of antibodies to (EENV)4 in Group 2 animals was higher than that found in the control (Group 4) animals.
In 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.
The serological analyses described above indicate that short repetitive sequences in RESA encode epitopes that are targets of host-protective antibodies. Thus, synthetic peptides corresponding to these sequences (or multimers of them) together with an appropriate carrier should be sufficient to immunize against the blood-stages of P.falciparum. EXAMPLE 35 - Immunogenicity of synthetic peptides. (a) Immunogenicity in Mice. The immunogenicity of synthetic peptides corresponding to RESA repetitive sequences has been demonstrated by immunizing mice with peptides conjugated to keyhole limpet haemocyanin (KLH) . These experiments used the same two RESA 3' repeat peptides as were used in the serological analysis of the Aotus vaccine trial i.e. (EENV)4 and EENVEHDA. The third peptide used in these immunogenicity tests was 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).
Sera from 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) . In addition 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 reverse, however, was not true: antibodies raised to EENVEHDA did not react with (EENV)4. When these anti-peptide antisera were assayed on peptide-BSA conjugates there was no apparent cross-reactivity between the 5' and 3' repeats of RESA. However, assay of the same sera on fused polypeptides revealed that the peptides had induced antibodies that reacted with both repeat structures, although the reaction with the heterologous repeat was very weak in comparison to that with the homologous repeat.
When these anti-peptide antisera were assayed on sonicates of erythrocytes infected with P.falciparum (Figure 24) the strongest signals were obtained from mice immunized with, (EENV)4 with all five mice in this group giving signals well above background when assayed at a dilution of 1:250. In contrast two of three mice immunized with DDEHVEEPTVAY, and 1 of 4 mice immunized with EENVEHDA gave signals above background. Despite these relatively low signals it is clear that each of these synthetic peptides is capable of inducing antibody responses that not only react with the immunizing peptide but also with the parasite antigen containing these sequences.
(b) Immunogenicity in rabbits. 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 250μg 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.
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.
TABLE 3 ANTIBODY RESPONSES IN RABBITS TO RESA SYNTHETIC PEPTIDES CONJUGATED TO DIPHTHERIA TOXOID.
* Antibody Respons es to :
Peptide Adjuvant
8 x 4mer 4 x 8-mer 3 x 11-mer
3 x 11-mer FCA 0.22 < 0.1 2.17
MDP < 0 .1 < 0.1 2.17
Alum < 0.1 < 0.1 0 .42
4 x 8-mer FCA < 0.1 2.10 < 0.1
MDP 0.26 1.85 < 0.1
Alum < 0 .1 < 0 .1 < 0 .1
8 x 4-mer MDP 1.28 < 0 .1 < 0.1
These values are the optical densities at 414nm assaying 1:1000 dilutions of rabbit sera by micro ELISA. Each value is the average for three rabbits.
INDUSTRIAL APPLICATION 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. REFERENCES
1. Atherton E., et al., (1983), J.Chem.Soc.Perkin Trans. I, 1:65-73.
2. Cowman, A.F. et.al. (1984), Molec. Biol.Med. , 2:207-221.
3. Kemp, D.J., et al., (1983), Proc.Natl.Acad.Sci.USA, 80:3787-3791
4. Merrifield, D.B. (1963) J.Am.Chem. Soc. 85:2149-2154.
5. Sanger, F. , et.al. (1977) Proc.Natl.Acad.Sci. USA, 74:5463-5467.
6. Willson, T.A., et.al. (1985) Gene Anal.Techn. 2:77-82.
7. Zoller, M.J. and Smith, M. , (1983) Methods Enzymol. 100B:468-500.

Claims

CLAIMS :
1. A polynucleotide sequence which has been derived from regions coding for the RESA antigen of Plasmodium falciparum, a polynucleotide sequence which hybridizes to said first-mentioned polynucleotide sequence, a polynucleotide sequence related to said first-mentioned sequence or said hybridizing sequence, or a polynucleotide sequence which on expression codes for a polypeptide derived from the RESA antigen of Plasmodium falciparum or which displays similar biological or immunological activity to said polypeptide.
2. A polynucleotide sequence which has been derived from the 5' and/or 3' repeat region of the RESA antigen of Plasmodium falciparum, a polynucleotide sequence which hybridizes to a said first-mentioned polynucleotide sequence, a polynucleotide sequence, a polynucleotide sequence related to said first-mentioned sequence or said hybridizing sequence, or a polynucleotide sequence which on expression codes for a polypeptide derived from the 5' and/or 3' repeat region of the RESA antigen of Plasmodium falciparum or which displays similar biological or immunological activity to said polypeptide.
3. A polynucleotide sequence which is a part, analogue, homologue, derivative or combination, or a multimer of all or parts, analogies, homologues, derivatives or combinations of at least one polynucleotide sequence according to claim 1 or claim 2.
4. A probe useful for identifying a polynucleotide sequence according to any one of claims 1 to 3 , which comprises a nucleotide sequence derived from regions coding for the RESA antigen of Plasmodium falciparum or which codes for a polypeptide displaying sijmilar biological or immunological activity to regions of the RESA antigen of Plasmodium falciparum or a sequence which hybridizes to said nucleotide sequence, and a label.
5. A recombinant DNA molecule, characterized by a DNA insert comprising a polynucleotide sequence according to any one of claims 1 to 3 , and vector DNA.
6. A recombinant DNA molecule according to claim 5 , wherein the vector DNA comprises plasmid, virus or bacteriophage DNA.
7. A recombinant DNA molecule according to claim 5 or claim 6, having an expression control sequence is operatively linked thereto.
8. A transformant host, wherein said host is transformed with a recombinant DNA molecule according to any one of claims 5 to 7.
9. A transformant host according to claim 8 , wherein the host cells are selected from bacterial, yeast, fungal and higher eukaryotic cells.
10. An expression product of a transformant host according to claim 8 or claim 9.
11. An expression product according to claim 10 in substantially pure form.
12. A polypeptide comprising a monomer or multimer of fragments of the RESA antigen of Plasmodium falciparum, or parts, analogues, homologues, derivatives or combinations of said fragments, or a polypeptide displaying similar biological or immunological properties to said first-mentioned polypeptide.
13. A polypeptide according to claim 12 , wherein said fragments comprise the 5' and/or 3' repeat unit(s) of the RESA antigen of Plasmodium falciparum.
14. A composition for stimulating immune responses in a mammal, which comprises at least one polypeptide according to claim 12 or claim 13, either alone or together with a pharmaceutically acceptable carrier and/or adjuvant.
15. A method of providing immunity in a mammal, which comprises administering to the mammal an effective amount of a composition according to claim 14.
16. A method for detecting anti-malarial antibodies, which comprises preparing a reagent comprising at least one polypeptide according to claim 12 or claim 13 , and employing said reagent in an assay to detect anti-malarial antibodies.
17. Antibodies raised against a polypeptide according to claim 12 or claim 13.
18. A method for detecting malarial antigens, which comprises preparing a reagent comprising antibodies according to claim 17, and employing said reagent in an assay to detect malarial antigens.
19. A composition for immunization against blood stage P.falciparum antigens in a mammal, comprising a synthetic peptide having or including a. least one sequence selected from the group consisting of: (DDEHVEEPTVA)n (EENVEHDA) n and (EENV) n wherein n is a positive integer, or a related sequence derived therefrom by deletion and/or conservative substitution, said synthetic peptide optionally being coupled to a carrier molecule, together with a pharmaceutically acceptable diluent and/or adjuvant.
20. A composition according to claim 19 , wherein said synthetic peptide has or includes at least one sequence selected from (DDEHVEEPTVA)n and (EENVEHDA) n, wherein n is a positive integer.
EP19870902028 1986-03-14 1987-03-13 Polypeptides providing protective immunity against malaria. Withdrawn EP0297110A4 (en)

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IT1213576B (en) * 1986-12-23 1989-12-20 Eniricerche Spa POLYPEPTID COMPOSITION USEFUL FOR THE PREPARATION OF ANIMALARY VACCINES AND DIAGNOSTIC KITS FOR THE DETERMINATION OF ANTI-MEROZOITE ANTIBODIES.
FR2672290B1 (en) * 1991-02-05 1995-04-21 Pasteur Institut SPECIFIC PEPTIDE SEQUENCES OF THE HEPATIC STAGES OF P. FALCIPARUM CARRIERS OF EPITOPES CAPABLE OF STIMULATING T-LYMPHOCYTES
CA2114223A1 (en) * 1991-07-31 1993-02-18 Denise Mattei Polypeptides capable of in vivo induction of antibodies themselves capable of inhibiting the invasion of red blood corpuscles by merozoites of p. falciparum, related products and their use in producing vaccine compositions

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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

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Biotechnology, Vol. 3, No. 8, August 1985, pages 729-740, New York, US; J.V. RAVETCH et al.: "Molecular genetic strategies for the development of anti-malaraial vaccines", page 733, column 2, lines 16-45; page 736, table 2. *
CHEMICAL ABSTRACTS, Vol. 105, No. 19, November 10, 1986, page 552, Abstract 170174p, Columbus, Ohio, US; P. PERLMANN et al.: "Specificity and inhibitory activity of antibodies to a Plasmodium falciparum antigen (Pf 155) and its major amino acid repeat sequence", & Vaccines 86, New Approaches Immun., (Proc. Conf), 1985, (Publ. 1986), 149-155, Abstract. *
Nucleic Acids Research, Vol. 14, No. 21, 1986, pages 8265-8277, J.M. FAVALORO et al.: "Structure of the RESA gene of Plasmodium falciparum", whole document. *
Proc. Natl. Acad. Sci. USA, Vol. 84, No. 5, March 1987, pages 1399-1403, Washington, DC, US; L. ASLUND et al.: "Synthetic gene construct expressing a repeated and highly immunogenic epitope of the Plasmodium falciparum antigen Pf155", whole document. *
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See also references of WO8705607A1 *

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