EP0434751A1 - Antigene de malaria - Google Patents

Antigene de malaria

Info

Publication number
EP0434751A1
EP0434751A1 EP89911024A EP89911024A EP0434751A1 EP 0434751 A1 EP0434751 A1 EP 0434751A1 EP 89911024 A EP89911024 A EP 89911024A EP 89911024 A EP89911024 A EP 89911024A EP 0434751 A1 EP0434751 A1 EP 0434751A1
Authority
EP
European Patent Office
Prior art keywords
polypeptide
antibody
glu
glurp
vaccine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP89911024A
Other languages
German (de)
English (en)
Inventor
Morten Dziegiel
Martin Borre
Soren Jepsen
Jens Vuust
Klaus Rieneck
Anette Wind
Palle Hoy Jakobsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Statens Serum Institut SSI
Original Assignee
Statens Serum Institut SSI
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DK519188A external-priority patent/DK519188D0/da
Application filed by Statens Serum Institut SSI filed Critical Statens Serum Institut SSI
Publication of EP0434751A1 publication Critical patent/EP0434751A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • a malaria antigen A malaria antigen
  • the present invention relates to a polypeptide comprising a characteristic amino acid sequence derived from the Plasmodium falciparum antigen GLURP, a polypeptide which is recognized by an antibody raised against or reactive with a polypeptide comprising said characteristic amino acid sequence and/or an antibody reactive with native GLURP, a nucleic acid molecule encoding said polypeptide, an expression vector carrying the nucleic acid molecule an organism expressing said nucleic acid molecule so as to produce said
  • Plasmodium The species Plasmodium falciparum is the most dangerous and malignant malaria parasite, causing acute severe infections that are often fatal, especially in young children and immigrants entering endemic areas. Thus, it is very desirable that a vaccine against P . falciparum is developed.
  • the life cycle of P. falciparum includes different stages; in the first stage, the sporozoite stage, the parasite is brought into the blood stream by the Anopheles mosquito.
  • the sporozoites are carried in the blood stream to the liver where they invade the hepatocytes and develop into merozoites in the course of 5-7 days. Merozoites released from infected cells start a new cycle by invading the erythrocytes.
  • the parasite shows an asexual multiplication which involve a maturation of the parasite through different parasite stages, the ring, the trophozoite and the schizont stage (the stage that undergoes nuclear division).
  • the schizont infected erythrocyte bursts, new merozoites are released. It is the disintegration of the erythrocyte which gives rise to the clinical disease.
  • gametocytes microgametocytes and macrogametocytes
  • merozoites differentiate into gametocytes (microgametocytes and macrogametocytes), the sexual form of the parasite.
  • these sexual parasite stages are able to continue the life cycle, when the infected cells, the erythrocytes, are ingested by mosquitoes during a blood meal.
  • the gametocytes develop into a mobile ookinete stage.
  • the ookinete pass through the epithel and matures into a oocyst.
  • the new sporozoites develop.
  • the parasites are haploid in most of the life cyclus as they perform a meiotic cell division shortly after fertilization.
  • the Anopheles mosquito is the primary vector of malaria, but the disease may also be seen after blood transfusion, i.v. injections with contaminated equipment and after transfer from an infected mother to the newborn child through the placenta.
  • falciparum especially in the schizont stage , have been found in sera from infected individuals (1, 3, 4, 5, 6, 7), and plasma fractions containing these antigens have been isolated and described by Jepsen and Axelsen.
  • the antigens constitute a heterogeneous group of proteins and glycoproteins.
  • a mixture of soluble P. falciparum antigen (antigen 1 and antigen 2) have been isolated from in vitro grown P. falciparum (2).
  • references 1-7 None of the antigens 1-7 mentioned in references 1-7 have, however, separately been isolated and purified, and the antigens have only been characterized by reference to molecular weight, glycosylation and antigenicity, their amino acid composition and possible content of epitopes as well as the nucleic acid molecules encoding the antigens have not been mentioned or indicated.
  • nucleic acid sequences encoding polypeptides of various Plasmodium species have been isolated and analysed (8, 9, 10), but none of these nucleic acid sequences encode a polypeptide having a characteristic sequence GLURP and they have all been obtained following a strategy different from the one used for isolating the DNA-sequence encoding said characteristic amino acid sequence. This will be explained in details in the following.
  • EP 0 209 643 (Eniricerche S.p.A)
  • the present invention relates to a polypeptide comprising a characteristic amino acid sequence derived from the Plasmodium falciparum antigen GLURP, which comprises the following sequence:
  • characteristic amino acid sequence derived from the Plasmodium falciparum antigen GLURP is intended to mean an amino acid sequence, such as an epitope, which comprises amino acids constituting a substantially consecutive stretch (in terms of linear or spatial conformation) in GLURP, or amino acids found in a more or less non-consecutive conformation in GLURP, which amino acids constitute a secondary or tertiary conformation having interesting and useful properties, e.g. as immunogens.
  • amino acids present at different positions in GLURP but held together e.g. by chemical or physical bonds, e.g. by disulphide bridges, and thereby forming interesting tertiary configurations are to be understood as "characteristic amino acid sequences".
  • characteristic amino acid sequence may comprise a consecutive subsequence of the amino acid sequence of GLURP of greater or smaller length or a combination of two or more parts of such subsequences which may be separated by one or more amino acid sequences not related to GLURP.
  • the characteristic amino acid sequences may be directly bonded to each other.
  • epitope refers to a sequence or subsequence of the polypeptides of the invention or a derivative or an analogue thereof capable of stimulating or interacting with immunocompetent cells, especially epitopes against which antibodies showing desirable properties with regard to diagnosis, prophylaxis or treatment can be raised.
  • analogue is used in the present context to indicate a protein or polypeptide of a similar amino acid composition or sequence as the characteristic amino acid sequence derived from the P . falciparum antigen GLURP, allowing for minor variations which do not have an adverse effect on the immunogenicity of the analogue.
  • the analogous polypeptide or protein may be derived from a microorganism of another species than P . falciparum or may be partially or
  • the term is further intended to mean any immunogenic subsequence, functional equivalent or derivative of the characteristic amino acid sequence.
  • immunological subsequence is intended to indicate an amino acid sequence comprising at least one epitope reactive with an anti-GLURP antibody found in the serum of malaria-immune patients and/or eliciting antibodies which are reactive with native GLURP.
  • the term "functional equivalent” is intended to include all immunogenically active substances with the ability to evoke an immune response in an animal, including a human being, to which a vaccine containing the equivalent has been administered which is similar to the immune response evoked by the characteristic amino acid sequence of GLURP, e.g. an anti-ideotypic antibody, in that it is able to confer immunity to diseases caused by plasmodial parasites.
  • the functional equivalent may be derived from a microorganism of another species than P. falciparum or may partially or completely be of synthetic origin. It should be understood that the similarities between the characteristic amino acid sequence from GLURP and the functional equivalent are qualitative rather than quantitative, relating to the nature rather than the level of activity of the functional equivalent.
  • the present invention also relates to a naturally or non-naturally occuring polypeptide which comprises a least one epitope reactive with an antibody which recognizes the P. falciparum antigen GLURP.
  • said epitope can be a subsequence of the amino acid sequence of GLURP.
  • the polypeptide and the epitope can have an amino acid sequence substantially homologous with but not identical to the amino acid sequence of GLURP provided that said epitope is reactive with an antibody which recognizes the P . falciparum antigen GLURP.
  • the antibody used for the recognition of the polypeptide of the invention may be a monoclonal or polyclonal antibody, which have been raised specifically against the polypeptide comprising the amino acid sequence outlined above. Monoclonal or polyclonal antibodies useful for the recognition of the polypeptide of the invention as well as methods of their production are described below.
  • the antibody is obtained from serum obtained from malaria-immune patients, e.g. from the malaria-immune serum pool available from Statens Seruminstitut, Copenhagen, Denmark.
  • the antibody may be obtained from the serum by use of conventional methods, e.g. as described in Materials and Methods below.
  • crossed immunoelectrophoresis By the term “recognized” is meant that a reaction between the polypeptide of the invention and the antibodies is observed, when the polypeptide and the antibody is allowed to react under circumstances which allow for such an observation.
  • the reaction may be in the form of a precipitation.
  • An analysis based on the principle of crossed immunoelectrophoresis has been found to be useful in this respect.
  • the crossed immunoelectrophoresis may be carried out substantially as described in (1) and as illustrated in the following examples.
  • the invention in another aspect, relates to a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide described above.
  • the nucleic acid molecule may be used in a method of preparing the polypeptide by recombinant DNA techniques or as a diagnosti agent (i.e. a DNA probe).
  • a nucleic acid molecule of the invention in the production of a recombinant polypeptide (e.g. by inserting the fragment in a suitable vector, transforming the vector into a suitable host microorganism, cultivating the microorganism so as to produce the polypeptide and subsequently recovering the polypeptide from the microorganisms) includes a number of advantages.
  • nucleic acid molecule of the invention may also be used in a
  • diagnostic agent for the detection of P. falciparum nucleic acid molecule in a sample, which diagnostic agent comprises a labelled nucleic acid molecule which is substantially homologous with a nucleic acid molecule coding for at least part of said polypeptide.
  • the present invention relates to a vaccine for immunizing an animal, including a human being, against diseases caused by a plasmodial parasite, which vaccine comprises an immunologically effective and physiologically acceptable amount of a polypeptide as defined above together with a physiologically
  • the vaccine should be made so as to allow an optimal stimulation of the relevant parts of the immune system, i.e to present the immunogenic polypeptide for a period of time and in a form being optimal with respect to the recognization, the uptake or any other interaction or processing necessary for the stimulation.
  • the polypeptide of the invention may be used in the preparation of a monoclonal antibody which is reactive with non-carbohydrate moieties of the polypeptide.
  • the polypeptide or the antibody may be used for the identification and/or quantification of at least part of the above described polypeptide present in a sample thus making it possible to diagnose Plasmodium species-induced diseases.
  • the sample may be any part of a living organism such as a human or an animal containing Plasmodium species molecules, or a specimen obtained from said living organism.
  • the sample may e.g. be a body fluid or tissue part containing the polypeptide, e.g. a tissue sample such as a biopsy, e.g.
  • Plasmodium species molecules may also be water, such as tap water, or foodstuffs, such as meat, or may be a vaccine or diagnostic agent in which it is desirable to determine the presence and/or quantity of Plasmodium species molecules.
  • the Plasmodium species molecules to be identified or quantified may be Plasmodium species molecules present on or being a part of surfaces of cells or within the cells present in the sample.
  • Plasmodium species molecules designates any molecule, e.g. a polypeptide, present on cell surfaces or being part of cell surfaces of the Plasmodium species as well as any molecules present in the cells such as in the cytoplasm or in the nucleus. Further, Plasmodium species molecules designates molecules being "detached from” or “secreted by" Plasmodium species containing cells,
  • the polypeptide of the invention is as mentioned above related to the P. falciparum antigen GLURP.
  • the amino acid sequence outlined above constitutes GLURP and is deduced from a nucleic acid sequence which was isolated from a genomic library by screening clones of the library with human malaria-immune sera and with antibodies which had been affinity purified on native antigen 1, a P. falciparum protein found in serum of malaria-immune patients.
  • the antibodies which were used for the screening, were shown to interact with precipitates representing antigen 1 in a crossed immuno-electrophoresis, and were therefore believed to be
  • Clones in the library reacting with these antibodies were therefore presumed to contain DNA inserts encoding antigen 1 or a part thereof.
  • the fusion protein which was purified from one of clones was used for affinity purification of human antibodies specific for the fusion protein.
  • the source of antibodies was an immune Liberian individual possessing high antibody titre against several of the soluble antigens of P . falciparum, including antigen 1.
  • Antibodies purified using the fusion protein column were tested in crossed Immunoelectrophoresis. It was found that they interacted with all of the precipitates representing antigen 1.
  • the malaria-immune serum used for the second dimension electrophoresis probably did not contain a sufficient amount of antibodies directed against GLURP to produce a visible precipitate by itself, only a deposition of affinity purified antibodies In the intermediate gel caused a precipitation of GLURP. Furthermore, the presence of antibodies directed against antigen 1 in the eluate from the fusion protein column was probably caused by unspeclfic absorption (see Fig. 1).
  • Antigen 1 has not been isolated and purified from the malaria-immune serum and it was not possible to compare the polypeptide of the invention with antigen 1 on the amino acid level or compare the isolated polypeptide with an isolated antigen 1.
  • Polypeptides of the type described above comprising a considerable amount of hydrophilic and/or acidic amino acids have been found to be of particular interest, especially with regard to their immunogenic properties. It is believed that the hydrophilic and acidic nature of the amino acids is responsible for the establishment of a conformational structure, e.g. a tertiary structure, which is advantageous in exposing the antigenic determinants of the polypeptide and thereby the binding of the polypeptide to a suitable substance, e.g. an antibody, when the polypeptide comprises an antigenic determinant.
  • the considerable amount of hydrophilic and/or acidic amino acids may also be advantageous in the recognition of suitable substances, e.g. antibodies, by the polypeptide.
  • the polypeptide of the invention is substantially pure.
  • the term "substantially pure” is understood to mean that the polypeptide in question is substantially free from other components, e.g. other immunologically active components, which may result from the production and/or recovery of the polypeptide or otherwise be found together with the polypeptide.
  • the high purity of the polypeptide of the invention is advantageous when the polypeptide is to be used for immunization purposes, e.g. as a vaccine constituent, as unwanted and adverse immune reactions resulting from the presence of other immunogenic components is avoided.
  • the substantially pure polypeptide may be used In a lower amount than a polypeptide of a conventional lower purity for most purposes. Further, the immunogenic concentration and/or
  • composition (constituted of the polypeptide of the invention) which is used for a given immunization purpose, e.g. in the form of a vaccine, can be precisely determined.
  • the purity of the polypeptide of the invention can be determined by Western Blot analysis and visualization of the polypeptide by Coomasie brilliant blue staining which will be dealt with in further details in the following.
  • GLURP The sequence of GLURP outlined above is constituted of 783 amino acid residues, which correspond to a molecular weight of 90 kD.
  • the amount of hydrophilic amino acids as well as acidic amino acids Is high.
  • the high content of the amino acid glutamate is, however, not a unique feature of a malaria protein.
  • GLURP is an abbreviation for glutamate rich protein.
  • the terminology of the topic DNA and protein is as follows: The DNA-insert from the original ⁇ -phage-clone is termed glurp and the protein encoded hereof is termed GLURP.
  • the fusion protein encoded by the plasmid pRD15 consisting of the N-terminal part of the ⁇ Cro-protein, the N-terminal part of ⁇ -galactosidase and GLURP, is termed ⁇ -gal:: GLURP (see Fig. 2).
  • the protein from the malaria parasite of which GLURP is believed to be the C-terminal part is called native GLURP.
  • GLURP has the following amino acid composition:
  • the hydropathy of GLURP has been analyzed using the indexes of Kyte and Doolittle (Kyte and Doolittle. J. Mol. Biol, 157 : 105-132 , 1982) . This shows, that the protein consists of a major predominantly hydrophilic amino terminal part (approximately residue 1-734) and a minor predominantly hydrophobic carboxy terminal part (approximately residue 734-783).
  • the hydropathy is illustrated in Fig. 3.
  • GLURP has a unique primary structure consisting of 3 unique repeats and an interspersed non-repeated sequence. This is also evident from the DNA sequence encoding GLURP, which DNA sequence is illustrated in Fig. 7, and further described below.
  • the sequence of the first repeat comprises
  • the second repeat comprises
  • SEKSVSEPAEHVEIV ⁇
  • the amino acid sequence is amino terminal to the 11 repeats presumed to be involved in the interaction with glycophorin on the surface of the red blood cell (Kochan J, Perkins M.and Ravetch J V: Cell, 44; 689-696:1986).
  • the amino acid proline does not occupy position 3 of the substantially repeated subsequence.
  • the polypeptide of the invention is characterized by having a glutamic acid composition of at least 20% and at the most 1 methione residue and/or no cystein residues.
  • the polypeptide of the invention is preferably capable of inducing a proliferative response in a T-lymphocyte.
  • proliferative response is understood to mean that the T-lymphocyte responds to an exposure to a polypeptide of the invention, e.g. antigenic deter minant(s) presented by the polypeptide, by producing substances such as interferon and interleukin, which are capable of eliciting an antibody production in B-lymphocytes of the immune system.
  • the proliferative response may be determined by detecting and optionally quantifying the Interferon and interleukin produced upon the exposure or by allowing the interferon or interleukin produced to elicit the antibody production from B-lymphocytes and determining the presence and/or amount of the resulting antibodies.
  • a polypeptide (T-cell epitope) eliciting a proliferative response in T-lymphocytes may advantageously be used in combination with a polypeptide (B-cell epitope) being recognized by antibodies produced by the B-lymphocytes for immunization purposes.
  • B-cell epitope polypeptide
  • B-cell epitopes is meant the structures in the polypeptide which interact specifically with the variable part of an immunoglobulin and thus, the B-cell epitopes are recognized by antibodies produced by the B-cells.
  • the structure of the polypeptide constituting the B-cell epitope may be a stretch of amino acids in the primary sequence of the polypeptide or a group of amino acids which are brought spatially together from parts of the sequence which are not contiguous in the primary sequence, e.g. by means of the secondary or tertiary structure of the polypeptide.
  • B-cell epitopes contain a rather small number of amino acids, e.g. comprising from about 3 to about 20 amino acids, more usually from about 4 to about 12 amino acids.
  • T-cell epitopes is to be understood as the structures in the polypeptide which are presented by antigen-presenting cells and which interact with the T-cell receptor.
  • the interaction between the antigen-presenting cells i.e. macrophages, B-cells, dendritic cells, interdigitating cells and Langerhans cells
  • the antigen-presenting cells Internalize the antigens by endocytosis or pinocytosis and subsequent processing by proteolytic cleavage of the antigens to smaller fragments, which are subsequently transformed to the cell surface and presented to the T-cell whereby the interaction between the antigen-presenting cells and the T-cell receptor is established.
  • the processing has been shown to include proteolytic cleavage of the primary structure which produces fragments of the 8-20 amino acids having e.g. an amphifilic ⁇ -helical structure.
  • Other alternative ways of processing are evident as it has been shown that a T-cell epitope might be composed of non-contiguous amino acids of the primary structure of the polypeptide.
  • the amphifilic ⁇ -helices are then presented on the external cell surface in relation to the molecules of the major histocompatibility complex (class II).
  • T-cells specific for this antigen and triggers the production of lymfokines, growth factors, differentiation factors and some of the corresponding receptors.
  • These substances stimulate the B-cells to produce antibodies against B-cell epitopes related to the T-cell epitope and stimulates natural killer cells (NK cells), killer cells, macrophages and cytotoxic T-cell to engage targets presenting the antigen.
  • NK cells natural killer cells
  • the T-cell epitopes do not in themselves produce antibodies but elicit the antibody production from B-cells inter alia by producing interferon and interleukin which are involved in the stimulation of antibody production of the B-cells.
  • the T-cell epitopes are not necessarily recognized by the antibodies raised against GLURP.
  • the presence of T-cell epitopes may, however, be illustrated by their capability of inducing a proliferative response in T-cells, i.e. to induce production of interferon and interleukin.
  • the polypeptides of the invention may solely comprise T-cell epitopes or solely B-cell epitopes or a combination of these.
  • the composition of the polypeptides of the invention may be tailored to their intended use, e.g. their use as a vaccine component.
  • B-cell epitopes are advantageous for most applications as they are required for eliciting an antibody production.
  • T-cell epitopes are extremely advantageous as they enhance and accelerate the immune response and the production of antibodies. Furthermore, (NB se Credlt koncept) the memory function of the immune system resides in the T-cells. By stimulating this part of the immune system, the antibody production is significant after approximately 5 days. If the memory function is not participating, e.g. in the non-immunized animal, or if the antigen used for immunization does not contain a T-cell epitope, the antibody production is significant after several weeks, consisting primarily of low avidity IgM antibodies and after several months consisting of IgG antibodies of higher avidity.
  • amino acid sequences Illustrated below are contemplated to constitute suitable T-cell epitopes of GLURP.
  • the sequences have been found by computer analysis of the amino acid sequence illustrated above according to the AMPHI-program (Margalit, H, Spouge, J L, Cornette, J L, Cease, K B, Delisi, C and Berzofsky, J A:
  • Example 10 The AMPHI-program predicts sequences having an amphifilic ⁇ -helical structure. Several potential sequences have been found. Among these, the following sequences are estimated to be the most interesting using manual construction of helical wheels:
  • the polypeptide of the invention may be a fusion protein in which characteristic amino acid sequence(s) from GLURP is/are fused to a second amino acid sequence not derived from GLURP.
  • the amino acid sequence to which the characteristic amino acid sequence(s) from GLURP is/are fused may be one which results in an increased
  • an amino acid sequence which modifies, e.g. increases the immunogenicity may advantageously be coupled to one or more characteristic amino acid sequences from GLURP so as to adapt the resulting fusion protein for vaccine components.
  • the fusion protein may comprise ⁇ -galactosidase or a part thereof, e.g. the cro- ⁇ -galactosidase.
  • a fusion protein may comprise ⁇ -galactosidase or a part thereof, e.g. the cro- ⁇ -galactosidase.
  • cro- ⁇ -galactosidase is the fusion protein produced by the E. coli strain POP 2136 harboring the plasmid pRD15 encoding the fusion protein.
  • This E. coli strain has been deposited with Deutsche Sammlung von Mikroorganismen, DSM, in accordance with the Budapest Treaty on September, 15, 1988 under the Accession No . DSM 4815 .
  • the construct of the plasmid harbored in the deposited strain is illustrated on Fig. 2.
  • the production and characterization of the fusion protein are given in the following Examples and illustrated in the drawings.
  • the characteristic amino acid sequence(s) from GLURP is/are
  • cleaving agent e.g. a chemical such as cyanogen bromide, hydroxylamine and 2-nitro-5-thiocyanobenzoate
  • an enzyme e.g. a peptidase, proteinase or protease, e.g. trypsin, chlostripain, and staphyllococal protease.
  • polypeptide of the invention may be coupled to a carbohydrate or a lipid moiety, e.g. a carrier, or modified in other ways, e.g. being acetylated.
  • a carbohydrate or a lipid moiety e.g. a carrier
  • the polypeptide of the invention will normally not be acetylated if no special measures are taken.
  • the acetylation may be advantageous as acetylated polypeptides may be more stable in cell, blood or body and tissue fluids.
  • the acetylation may confer the polypeptide with a structure and conformation which mimics the structure and confirmation of the native P . falciparum antigen GLURP.
  • Example 12 it is demonstrated that the polypeptide of the invention can stimulate the immune system without being modified.
  • the polypeptide of the invention is In a non-glycosylated form.
  • the polypeptide of the invention is derived from a
  • Plasmodium species preferably P. falciparum.
  • the Plasmodium species preferably P. falciparum.
  • Plasmodium species, from which the polypeptide is derived, is in the schizont stage.
  • polypeptide of the invention is contemplated to be useful as a prophylactic or therapeutic agent, e.g. a vaccine, or in a diagnostic kit and may be used in the manufacture thereof. This will be explained below.
  • nucleic acid molecule encoding the polypeptide of the invention.
  • the invention relates to a nucleic acid molecule comprising substantially the following nucleotide sequence: 1 GAATTCGTTG AATCGGAAAA AAGCGAGCAT GAAGCAGCTG AAAATGAAGA AAGTAGTCTT
  • A represents adenine
  • T represents thymidine
  • G represents guanine
  • C represents cytosine
  • this nucleotide sequence encodes the carboxylic terminal part of native GLURP, due to the fact that an open reading frame of 2349 basepairs extends from the 5'-terminal end of the insert to a "TAA" stop codon (indicated by an arrow in the table above). This is the longest open reading frame found in the nucleotide sequence. No start or initiation codon appears in the reading frame indicating that the above sequence is the 3 '-part of the DNA-sequence encoding the carboxyl terminal end of native GLURP.
  • the DNA-sequence shown above has been established as described in the following examples.
  • Example 2 the form in which the protein encoded by the above nucleotide sequence was obtained, was as a fusion protein, containing most of the ⁇ -galactosidase peptide sequence at the amino terminal end.
  • the nucleic acid sequence encoding GLURP is presumed to terminate at position 2349 where a stop codon is found (indicated by an arrow). The remaining part of the sequence is noncoding.
  • the fusion protein encoded by the above nucleotide sequence was tested with respect to its antigenic properties as described in Examples 3 and 4.
  • the nucleic acid sequence displays some of the characteristics of other malaria nucleic acid sequences : Tandemly repeated motifs, high AT content (Hyde, John E. and Sims, P. F. G.,1987, Gene (61) pp. 177- 187) and a corresponding preference for codons containing these bases, and a high content of codons for glutamate.
  • the repetitive regions are indicated in the homology matrix, fig. 9 , as lines of dots appearing parallel to the diagonal representing the homology of the sequence with itself.
  • the figure illustrates the three major regions of repetitive
  • one motif from bp 34 to bp 156 is repeated from bp 289 to bp 411; another motif from bp 477 to bp 521 is repeated tandemly twice from bp 522 to bp 566 and from bp 567 to bp 611; a third motif from bp 1174 to bp 1233 is repeated tandemly 11 times.
  • This last repetitious region consists of 3 60 bp repeats and 8 57 bp repeats differing only in the 3 bases GAT coding for the amino acid aspartate. This region is flanked to the 5' terminal of a degenerated 60 bp repeat.
  • the GC content of the coding part of the Insert (shown In Fig. 10) is on the average 30%, and of the non coding 3' terminal 11% in
  • Hybridization is a useful method to compare the homology of GLURP to the sequence of a given DNA-molecule.
  • Hybridization may be performed as follows: Pure DNA comprising the nucleic acid sequence encoding GLURP from the plasmid pRD15 in POP2136 is prepared using the large scale method described in Maniatis et al. op . cit . , page 86-96. More specifically, glurp is excised from the plasmid by digestion of the plasmid DNA with EcoRI. The insert Is then separated from the plasmid DNA by use of agarose gel electrophoresis. The insert is labelled by any labelling principle, such as the ones disclosed herein.
  • the foreign DNA to be examined is coupled to a matrix, e.g. a matrix, e.g. a matrix, e.g. a matrix, e.g. a matrix, e.g. a matrix, e.g. a matrix, e.g.
  • nitrocellulose filter The filter is subjected to a suitable treatment suited to the kind of matrix employed so as to couple the DNA to the matrix, in the case of a nitrocellulose filter e.g. by baking the filter at a temperature of 80°C for 2 hours.
  • the membrane is exposed to a prehybridization solution of a composition, at a temperature and for a period of time recommended suited to the membrane in question.
  • the membrane is then placed in the hybridization solution containing the labelled denatured DNA probe obtained from the pRD15 plasmid (glurp). Hybridization is preferably carried out over night at a suitable temperature.
  • the membrane is then washed and incubated with a volume of 50 ml 2xSSC at 65°C for 30 minutes.
  • the procedure is repeated once.
  • the membrane is then incubated in 15 ml 2xSSC containing 0.1% SDS. Incubation is performed at 65°C for 30 minutes. All incubations including prehybridization and washings are performed with gentle agitation.
  • the filter is air-dried and wrapped in a suitable plastic wrap (e.g. Saran Wrap), the filter is then applied to an x-ray film so as to obtain an auto-radiographic image. Exposition is preferably carried out at -70°C with
  • any hybridization of glurp to the DNA is an indication of similarity of the sequences of the two nucleic acid molecules, i.e. that the DNA is a nucleic acid molecule of the invention.
  • Another approach of determining similarity between DNA molecules is by determining the nucleotide sequence of the DNA molecule to be compared with glurp or a subsequence of glurp by conventional DNA sequencing analysis, and comparing the degree of homology with the chosen subsequence of glurp . In the same way the homology of a given DNA sequence to the complementary DNA-sequence of glurp can be determined.
  • a degree of homology of at least about 70%, e.g. at least about 80% such as at least about 95% is obtained.
  • the nucleic acid sequence of the invention may comprise a nucleic acid sequence fused to another nucleic acid sequence encoding a characteristic amino acid sequence with the purpose of producing a fused polypeptide, e.g. the fusion protein illustrated in the
  • the fused sequence may be inserted into an appropriate vector which is transformed into a suitable host microorganism.
  • the nucleic acid molecule of the invention may be inserted in the vector in frame with a nucleic acid sequence carried by the vector, which nucleic acid sequence encodes a suitable polypeptide.
  • the host microorganism is grown under conditions ensuring expression of the fused sequence after which the fused polypeptide may be recovered from the culture by physico-chemical procedures, and the fused polypeptide may be subjected to gel filtration and affinity
  • polypeptide of the invention and the polypeptide to which it is fused may be separated, for instance by suitable proteolytic cleavage, and the polypeptide of the invention may be recovered, e.g. by affinity purification or another suitable method.
  • nucleic acid molecule of the invention is substantially complementary to at least a substantial portion of the nucleic acid molecule of Fig. 7.
  • the DNA-fragment may also comprise a suitable nucleotide sequence controlling the expression and replication of the nucleic acid molecule.
  • the regulatory nucleotide sequence is conveniently a part of the expression vector used for the production of the polypeptides, when such a vector is employed.
  • the nucleic acid molecule of the invention preferably contains a considerable number of codons corresponding to hydrophilic and/or acidic amino acids, e.g. codons corresponding to the amino acids glutamic acid and aspartic acid.
  • the nucleic acid molecule described above may be obtained from
  • Plasmodium species parasites e.g. from chromosomal or genomic DNA or by reverse transcriptase producing cDNA.
  • the nucleic acid molecule from chromosomal or genomic DNA it is preferably derived directly from the parasite genome, e.g. by screening for genomic sequences, hybridizing to a DNA probe prepared on the basis of the full or partial nucleic acid sequence of glurp .
  • the DNA is of complementary DNA (cDNA) origin, it may be obtained by preparing a cDNA library on the basis of mRNA from cells producing
  • cDNA differs from genomic DNA in, e.g. that it lacks certain transcriptional control elements and introns which are non-coding sequences within the coding DNA sequence. These elements and introns are normally contained in the genomic DNA.
  • the nucleic acid molecule may also be of synthetic origin, i.e. prepared by conventional DNA synthesizing method, e.g. by using a nucleotide synthesizer. The nucleic acid molecule may also be produced using a combination of these methods.
  • the invention relates to an expression vector which is capable of replicating in a host organism and which carries a nucleic acid molecule as described above and is capable of expressing the polypeptides described above.
  • the expression vector is capable of replicating in a host organism and of expressing therein a polypeptide which comprises at least one epitope reactive with an antibody which recognizes the P . falciparum antigen GLURP, such as an expressed peptide which is substantially homologous with a substantial portion of the polypeptide, the amino acid sequence of which is shown in Fig. 8.
  • the vector may be any vector which may conveniently be subj ected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may either be one which is capable of autonomous replication, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, or one which is replicated with the host chromosome, such as a bacteriophage.
  • plasmids such as natural or synthetic plasmids, eg. plasmids related to pBR322 such as pEX 1-3, the pRIT-family, the pUC-family and the like, and viruses such as adenovirus, vaccinia, retrovirus, Baculo virus, Epstein-Barrvirus, SV40-related virus and bovine papilloma virus.
  • suitable bacteriophages include M13 and lambda.
  • the invention also relates to an organism which carries and is capable of expressing a nucleic acid molecule as defined above and which not in its native form expresses said nucleic acid molecule.
  • the nucleic acid molecule may be carried on a vector as described above or may be integrated in the genome of the organism. Examples of suitable organisms include microorganisms such as bacteria, e.g.
  • bacteria of the genus Bacillus e.g. B . subtilis , Escherichia , e.g. E. coli, or Salmonella; yeasts, fungi, protozoa, insect cells and higher eucaryotic organisms or cells including plant and mammalian cells.
  • higher organisms such as animals, e.g. sheep, cattle, goats, pigs, etc. is contemplated to be useful as host organisms for the production of the polypeptide of the invention.
  • the present Invention also relates to a method of producing the polypeptides described above.
  • the polypeptides are prepared using recombinant DNA-technology e.g. the methods disclosed in
  • the polypeptides may be produced by a method which comprises cultivating or breeding an organism carrying a DNA-fragment encoding a characteristic amino acid sequence from GLURP, e.g. the above described nucleic acid molecule, under conditions leading to expression of said nucleic acid molecule, and subsequently recovering the polypeptide from the organism.
  • the organism which is used for the production of the polypeptide may be a higher organism, e.g. an animal, or a lower organism, e.g. a microorganism.
  • the nucleic acid molecule encoding the characteristic amino acid sequence from GLURP should be introduced in the organism.
  • the nucleic acid molecule is inserted in an expression vector, e.g. a vector as defined above, which is subsequently introduced into the host organism.
  • the nucleic acid molecule may also be directly inserted in the genome of the host organism. The insertion of the nucleic acid molecule in the genome may be accomplished by use of a virus, such as a bacteriophage, carrying the nucleic acid molecule and being capable of mediating the insertion into the host organism genome.
  • nucleic acid molecule into an expression vector or into the genome of the host organism may be accomplished as described e.g. by Colbere-Garapin F. et al., J. Molec. Biol., 150; 1-14 (1981): A New Dominant Hybrid Selective Marker for Higher Eucaryotic Cells.
  • the nucleic acid molecule when using an expression vector for the production of the polypeptide of the invention, may be inserted in frame with a second nucleic acid molecule encoding another polypeptide so as to obtain an expression of fusion protein.
  • the polypeptide of the invention comprises one or more distinct parts, e.g. being a fusion protein comprising on the one hand characteristic amino acid sequence(s) from GLURP and on the other hand amino acid sequence(s) constituting a polypeptide which is not related to GLURP
  • the nucleic acid molecules encoding each of these polypeptides may be inserted in the genome or expression vector separately or may be coupled before insertion into the genome or expression vector by use of conventional DNA techniques such as described in Maniatis et al.
  • the conditions under which the organism producing the polypeptide of the invention is cultured or breeded should of course be adapted to the organism employed. Conventional cultivation and breeding techniques may be employed. In the case of microorganism, the cultivation is e.g. carried out in a culture medium conventionally used for fermentation purposes, e.g. Luria Broth medium, and under conditions with respect to pH, temperature, aeration, etc. suited to the type of microorganism in question, e.g. as disclosed in Maniatis et al. op . cit.
  • the polypeptide is recovered or isolated from the organism.
  • the polypeptide may be isolated or recovered from the culture by a method comprising one or more affinity chromatography and/or size chromatography steps, and optionally employing a step using an antibody reactive with and/or being raised against said polypeptide.
  • the procedure used for recovering of the polypeptide depends on the kind of host organism used as well as the polypeptide produced.
  • the recovery and isolation of the polypeptide will also of course depend on the kind of microorganism employed.
  • the recovering of the polypeptide from the microorganism comprises treatment of the microorganism so as to release the polypeptide, e.g. by rupturing the microorganism, i.e. partly or totally, and subsequently recovering the polypeptide by well-known methods such as precipitation, gel filtration, ion exchange chromatography, or HPLC reverse phase chromatography or immuno affinity chromatography or the like.
  • the polypeptide of the invention may be isolated from a biological material containing the polypeptide, e.g. a suspension of cells producing the polypeptide, by use of a method comprising adsorbing the biological material to a matrix comprising an immobilized monoclonal or polyclonal antibody as described herein, eluting the polypeptide from the matrix, and recovering the polypeptide from the eluate.
  • a biological material containing the polypeptide e.g. a suspension of cells producing the polypeptide
  • a method comprising adsorbing the biological material to a matrix comprising an immobilized monoclonal or polyclonal antibody as described herein, eluting the polypeptide from the matrix, and recovering the polypeptide from the eluate.
  • procedures for isolating the polypeptide are: a) A procedure employing antibodies reactive with Plasmodium species molecules which is suited for the obtainment of a Plasmodium species containing fraction with high purity, especially a fraction which contains molecules
  • the procedure may be performed by immobilizing the specific antibodies, preferably monoclonal antibodies, to a matrix, contacting said matrix with the preparation containing the released Plasmodium species molecules, washing, and finally treating the antigen-antibody complex fixed to the matrix so as to release the Plasmodium species molecules in a purified form.
  • a preferred way is to isolate the Plasmodium species molecules by means of column affinity chromatography involving antibodies fixed to the column matrix. b) Procedures involving various forms of affinity chromatography, gel filtration, ion exchange or high performance liquid chromatography (HPLC).
  • Preparative electrophoresis procedures for instance the following procedure: A supernatant from a centrifuged enzyme treated cell or cell line preparation is subjected to a gel electrophoresis, such as a sodium dodecyl sulphate-polyacrylamidgel electrophoresis
  • labelled antibodies such as monoclonal antibodies, reactive with Plasmodium species, are used to Identify bands primarily constituted by the isolated Plasmodium species molecules.
  • the antibodies may be used in any conventional immunoblotting technique.
  • the markers may be isotopes or fluorescein labels detectable by means of relevant sensitive films.
  • the Plasmodium species containing bands of the gel may be subjected to a treatment resulting in the release of the Plasmodium species molecules from the gels, such as procedures involving slicing up the gel and subsequent elution of Plasmodium species molecules.
  • a treatment resulting in the release of the Plasmodium species molecules from the gels such as procedures involving slicing up the gel and subsequent elution of Plasmodium species molecules.
  • Plasmodium species proteins obtained may be determined. Prior to cultivation of the microorganism, the nucleic acid molecule encoding the polypeptide of the invention may be subjected to modification, before or after the nucleic acid molecule has been inserted in the vector. The polypeptide produced may also be subjected to modification.
  • the modification may comprise
  • substitution is intended to mean the replacement of any one or more amino acids or nucleotides in the full amino acid or nucleotide sequence with one or more others
  • addition is understood to mean the addition of one or more amino acids or nucleotides at either end of the full amino acid or nucleotide sequence
  • insertion is intended to mean the introduction of one or more amino acids or nucleotides within the full amino acid or nucleotide sequence
  • detion is intended to indicate that one or more amino acids or nucleotides have been deleted from the full amino acid or nucleotide sequence whether at either end of the sequence or at any suitable point within it.
  • nucleic acid molecule may, however, also be modified by subjecting the organism carrying the nucleic acid molecule to mutagenization, preferably site directed mutagenization so as to mutagenize said fragment.
  • mutagenization may be performed by using conventional mutagenization means such as ultraviolet
  • nucleotide substitutions which do not give rise to another amino acid sequence of the protein, but which, e.g., correspond to the codon usage of the specific organism in which the sequence is inserted; nucleotide substitutions which give rise to a different amino acid sequence and therefore, possibly, a different protein structure without, however, impairing the critical properties of the polypeptide encoded by the DNA sequence; a subsequence of the DNA sequence shown above encoding a polypeptide which has retained the immunogenic properties of the native GLURP; or a DNA molecule hybridizing to at least part of a DNA molecule prepared on the basis of the DNA sequence shown above, provided that it encodes a polypeptide which has the biological property of native GLURP.
  • polypeptide produced as described above may be subjected to posttranslational modifications such as for instance thermal treatment, treatment with a chemical such as formaldehyde, glutar aldehyde or a suitable proteolytic enzyme, e.g. a peptidase or proteinase, such as trypsin, and substitution, addition, insertion, deletion, or rearrangement of one or more amino acids in the polypeptide.
  • posttranslational modifications such as for instance thermal treatment, treatment with a chemical such as formaldehyde, glutar aldehyde or a suitable proteolytic enzyme, e.g. a peptidase or proteinase, such as trypsin, and substitution, addition, insertion, deletion, or rearrangement of one or more amino acids in the polypeptide.
  • the posttranslational modification of the polypeptide may serve the purpose of adapting the polypeptide to a specific use, e.g. as a component in the vaccine such as described herein.
  • the lack of glycosylation of the polypeptide produced by the E. coli strain DSM 4815 has been found not to affect the immunogenic and antigenic properties ⁇ -gal::GLURP (see Example 3, 4 and 12) in any substantial manner; the polypeptide shows the characteristic reaction, e.g. it precipitates with antibodies obtained from serum from malaria-immune patients. This is illustrated in the following examples. However, it may be
  • the polypeptide of the invention may be prepared by the well- known methods of liquid or solid phase peptide synthesis utilizing the successive coupling of the individual amino acids of the polypeptide sequence or the coupling of individual amino acids forming fragments of the polypeptide sequence which fragments subsequently are coupled so as to result in the desired polypeptide.
  • the solid phase peptide synthesis may e.g. be performed as described by R. B. Merrifield, J. Am. Chem. Soc . 85 , 1963, p. 2149.
  • the amino acid sequence is constructed by coupling an initial amino acid to a solid support and then sequentially adding the other amino acids in the sequence by peptide bonding until the desired length has been obtained.
  • the solid support may also serve as the carrier for the polypeptide of the invention in a vaccine preparation as described below.
  • the preparation of synthetic peptides for use as vaccines or for diagnostic purposes may be carried out essentially as described in Shinnick, Ann . Rev. Microbiol . 37, 1983, pp. 425-446.
  • the present invention also relates to a vaccine for immunizing an animal, including a human being, against diseases caused by a plasmodial parasite, which vaccine comprises an immunologically effective and physiologically acceptable amount of the polypeptide of the type described above together with a physiologically compatible carrier.
  • the term "vaccine” is to be understood to comprise any preparation containing an immunologically effective part of Plasmodium species molecules suited for administration to living organisms for the prevention, amelioration or treatment of Plasmodium species infection.
  • the plasmodial parasite is a P. falciparum.
  • the term “immunization” is understood to comprise the process of evoking a specific immunologic response with the expectation that this will result In humoral, and/or secretory, and/or cell-mediated immunity to infection with Plasmodium species, i.e. immunity is to be understood to comprise the ability of the individual to resist or overcome infection or to overcome infection "more easily” compared to individuals who have not been immunized or to tolerate the infection without being clinically affected or to block transmission.
  • the immunization according to the present invention is a process of increasing resistance to infection with Plasmodium. species.
  • An overall aspect in the preparation of the vaccines of the invention is the physiological acceptability of the components and of the total composition of the vaccine.
  • the final formulation of the vaccine should be a mixture of substances supporting and enhancing the immune response induced by the specific immunogenic component.
  • the vaccines of the present invention may suitably be provided as a sporozoite vaccine, merozoite and/or gamete vaccine.
  • the terms refer to the various stages of the life cycle of the malaria parasite described above. These stages may be targeted for immunological attack by a vaccine.
  • a vaccine which is strain-non-specific, i.e. it comprises an epitope which is a protective epitope common to substantially all strains of the
  • Plasmodium species causing infections of considerable clinical importance In this case, an epitope according to the present invention being conserved in different Plasmodium species is advantageous.
  • a multivalent vaccine is formulated, i.e. several immunologically effective components are incorporated into a single vaccine being effective in reducing infection, and/or transmission - all in all inducing an effective protective immunity.
  • the vaccine may comprise one or more additional molecules which are not related to GLURP in order to provide the multivalent nature of the vaccine.
  • additional molecules are immunologically active molecules obtained from pathogenic organisms other than Plasmodium species organisms which give rise to a vaccine being effective in reducing infection or providing immunity for one or more pathogenic organisms in addition to the plasmodial parasite.
  • cloned DNA sequences can be used for the synthesis of proteins and peptides.
  • a major advantage of this strategy is the ability to produce an unlimited amount of a purified product and the avoidance of contamination by pathogens.
  • Production can be carried out as described above, e.g. in a microorganism such as in bacteria or in yeast. Alternatively, liquid or solid phase synthesis can be used.
  • Routine methods for vaccine production involve risks of obtaining unwanted side effects, e.g. due to the vaccine containing unwanted (or even unidentified) contaminants.
  • the methods of preparation of vaccines according Ho the present invention are designed to ensure that the identity and immunological effectiveness of the specific molecules are maintained and that no unwanted microbial contaminants are introduced.
  • the final products are distributed under aseptic conditions into preferably sterile containers which are then sealed to exclude extraneous microorganisms.
  • the vaccine may further comprise art adjuvant in order to increase the immunogenicity of the vaccine preparation.
  • the adjuvant may serve the purpose of enhancing the stimulatory properties of the polypeptide by stimulating the production of cytokines or lymphokines from the cells of the immune system in a non-specific way.
  • the adjuvant may be selected from the group consisting of Freund's incomplete adjuvant (see Examples 12A and 12C), aluminium hydroxide (see Examples 12B and 12C), a saponin, a muramyl dipeptide, a lipopolysaccharide, a T-cell immunogen, inerleukin-2, interferon-gamma, an oil, such as a
  • Another vaccine form is contemplated to be useful as it improves the transportation of the vaccine and the physical-chemical presentation, and prolongs the time of presentation for the relevant parts of the immune system.
  • Such vaccine comprises a vehicle which may be in various forms.
  • the vaccine may comprise polypeptides incorporated into micelles, (using micelle-forming agents such as detergents, preferably non-ionic detergents or other non-denaturating micelle- forming agents such as amphiphilic peptides, glycosldes), open spherical structures, consisting of circular subunits or parts of spherical structures , the formation of which utilizes the hydro- phobic/hydrophilic properties of the polypeptides.
  • vaccines are contemplated in which the polypeptides are incorporated into so- called iscoms (immune stimulating complexes, as disclosed, e.g., in EP 0 109 942).
  • the polypeptide of the invention may advantageously be coupled to a carrier, which may be any carrier usually employed in the preparation of vaccines.
  • the carrier may be a macromolecular carrier, e.g.
  • the carrier should preferably be non-toxic and non-allergenie.
  • the polypeptide may be multivalently coupled to the macromolecular carrier as this provides an increased immunogenicity of the vaccine preparation.
  • it may prove advantageous to couple the polypeptide to the carrier together with one or more immunologically active molecules obtained from organisms other than plasmodium species so as to obtain a vaccine comprising a variety of different immunogenic determinants, i.e. a cocktail vaccine, which may be employed for the immunization of diseases caused by a variety of different organisms.
  • a vaccine wherein the polypeptide is polymerized, i.e. so as to present the polypeptide in a multivalent form, may also prove advantageous.
  • immunization schedules may be employed when using the vaccine of the invention: In some instances it may be appropriate to provide active immunization early in life. Furthermore, it may be desirable to employ repeated administrations, e.g. at regular or prolonged intervals, optionally - as far as injections are concerned - at various body sites, e.g. at the same time. Any immunization schedule which may be contemplated or shown to produce an appropriate immune response can be employed in accordance with the principles of the present invention.
  • the vaccine should be administered in a way which ensures an efficient stimulation of the immune system. This means that the vaccine should be brought into contact with the cells of the immune system for a sufficient period of time and in a form capable of functioning as an immunogen.
  • Several ways are possible. Of these the most conventional are the parenteral ways, i.e., the subcutaneous, intradermal, intramuscular or the intravenous route.
  • the aerosol vaccine is in most cases administered via the nasal route. It is known that peptides can be transported intactly through the nasal mucosa to reach the blood. When transported further down the respiratory tract, the antigen is taken up by the macrophages functioning as scavengers and is in this way potentially presented to the immune system. Some of the material administered as an aerosol may possibly reach the intestines and stimulate the immune system present in the intestines and this way stimulate the immune system of the body, or may be taken up by the intestinal mucosa in intact form and liberated to the blood stream where it will be presented for the immune system.
  • the vaccine may also be administered strictly via the nasal route. This way simplifies the administration and circumvents the problems associated with spreading of infectious diseases through multiple use of syringes.
  • the oral route of administrating the vaccine utilizes the finding, that certain proteins are taken up by the intestinal mucosa and are found in intact form in the bloodstream. This special way of
  • administering the vaccine will take advantage of pharmaceutical formulations protecting the immunogenic components from degradation In the stomach or in the intestines.
  • An effect of administrating the vaccine via the oral route may also come from the polypeptide stimulating that part of the immune system which is residing in the intestines and in the liver - and this way leading to a general immune stimulation.
  • the rectal route of administering the vaccine has the same advantages as the above mentioned methods but might be more reliable and thus lead to greater patient compliance in special groups, i.e. children.
  • the present invention relates to a non-pathogenic microorganism which carries and is capable of
  • a live vaccine for use as a live vaccine for the immunization of an animal, including a human being, against diseases caused by malaria parasites.
  • the use of a live vaccine might be advantageous since there is some indication that vaccines based on living organisms show excellent immunogenicity, often conferring a lifelong immunity against the disease in question. Live vaccines also tend to be less expensive to produce than those based on a purified protein, no purification step being required.
  • the polypeptide of the invention may advantageously be expressed on the outer surface of the non- pathogenic organism. This provides a favorable presentation of the polypeptide which will be recognized by the immune defense mechanisms of the animal to which the live vaccine is administered, thus provoking an appropriate immune response.
  • the vaccine as a recombinant organism, i.e. a bacteria such as of the genus Escherichia or
  • Salmonella this route could allow the bacteria to become established in the intestines and/or in the liver - and thus provide the patient with a prolonged immune stimulation.
  • one or more DNA sequences encoding antigens could be inserted into a virus genome, e.g. into a retrovirus, vaccinia, Epstein-Barr virus genome, to produce a polyvalent vaccine.
  • a DNA sequence encoding for a characteristic amino acid sequence related to Plasmodium species molecules and/or an immunologically equivalent or derivative thereof could be recombined with vaccinia to yield a vaccine to protect against infection with Plasmodium species.
  • passive immunization is employed, i.e. a preparation containing antibodies, e.g. of the type described below, especially a preparation with a high content of purified antibodies, is administered.
  • a mixture of two or more single vaccines is employed.
  • Another aspect of the invention is a monoclonal or polyclonal antibody specific for a Plasmodium species molecule such as the P . falciparum antigen GLURP or a polypeptide as described above, and a method for the preparation thereof .
  • the term "antibody” may refer to a substance which is formed by an animal or animal cell belonging to the immune system as a response to exposure to the polypeptides of the invention.
  • the variant domain of an antibody is composed of variable and constant sequences.
  • the variant part of the domain is called the idiotype of the antibody. This part of the antibody is responsible for the interaction with the antigen, the antigen binding.
  • the idiotypic structure is antigenic and can thus give rise to specific antibodies directed against the idiotypic structure. This has been done in mice.
  • the antibodies raised against the idiotype, the anti-idiotypic antibodies may mimic the structure of the original antigen and therefore may function as the original antigen to raise antibodies reactive with the original antigen.
  • This approach may be advantageous as it circumvents the problem associated with the characterization and synthesis of the important immunogenic parts of the protein in question. This is most important in the case of conformational epitopes, which might otherwise be difficult to identify. It has been shown for a number of organisms that protective immunity can be induced in this way (e.g. Trypanosoma druzei ,
  • the antibodies of the present invention may be produced by a method which comprises administering in an immunogenic form at least a part of the polypeptide of the invention to obtain cells producing antibodies reactive with said polypeptide and isolating the antibody containing material from the organism or the cells.
  • the methods of producing antibodies of the invention will be explained further below.
  • the antibody is preferably a monospecific antibody.
  • the monospecific antibody may be prepared by injecting a suitable animal with a substantially pure preparation of the polypeptide of the invention followed by one or more booster injections at suitable intervals (e.g. one or two weeks to a month) up to four or five months before the first bleeding.
  • suitable intervals e.g. one or two weeks to a month
  • the antibody may be a polyclonal antibody.
  • Polyclonal antibodies may be obtained, e.g. as described in Harboe and Ingild, see above. More specifically, when polyclonal antibodies are to be obtained, the Plasmodium species molecule preparation is, preferably after addition of a suitable adjuvant, such as Freund's incomplete or complete adjuvant, injected into an animal. When the immunogens are human Plasmodium species molecules, the animals may be rabbits.
  • the animals are bled regularly, for Instance at weekly Intervals, and the blood obtained is separated into an antibody containing serum fraction, and optionally said fraction is subjected to further conventional procedures for antibody purification, and/or procedures involving use of purified Plasmodium species molecules.
  • monoclonal antibodies are obtained.
  • the monoclonal antibody may be raised against or directed substantially against an essential component of Plasmodium species molecules, i.e. an epitope.
  • the monoclonal antibody may be produced by conventional techniques (e.g. as described by Köhler and Milstein,
  • the monoclonal antibody may be produced by fusing cells producing the monoclonal antibody with cells of a suitable cell line, and selecting and cloning the resulting hybridoma cells producing said monoclonal antibody.
  • the monoclonal antibody may be produced by immortalizing an unfused cell line producing said monoclonal antibody, subsequently growing the cells in a suitable medium to produce said antibody, and harvesting the monoclonal antibody from the growth medium.
  • the immunized animal used for the preparation of antibodies of the invention is preferably selected from the group consisting of rabbit, monkey, sheep, goat, mouse, rat, pig, horse and guinea pigs.
  • the cells producing the antibodies of the invention may be spleen cells or lymph cells, e.g. peripheral lymphocytes.
  • hybridoma cells When hybridoma cells are used in the production of antibodies of the invention, these may be grown in vitro or in a body cavity of an animal.
  • the antibody-producing cell is injected into an animal such as a mouse resulting in the formation of an ascites tumour which releases high concentrations of the antibody in the ascites of the animal.
  • the animals will also produce normal antibodies, these will only amount to a minor percentage of the monoclonal antibodies which may be purified from ascites by standard purification procedures such as centrifugation, filtration, precipitation, chromatography or a combination thereof.
  • An example of a suitable manner in which the monoclonal antibody may be produced is as a result of fusing spleen cells from immunized mice (such as Balb/c mice) with myeloma cells using conventional techni ques (e.g. as described by R. Dalchau, J. Kirkley, J.W. Fabre.
  • An especially interesting antibody Is a monoclonal antibody which is reactive with at least a part of the polypeptide encoded by the nucleic acid molecule of the invention and expressed from the microorganism deposited under the accession No. DSM 4815.
  • the invention relates to a diagnostic agent which comprises an antibody as defined above, preferably a monoclonal antibody.
  • the diagnostic agent may be in the form of a test kit comprising in a container a polypeptide comprising a characteristic amino acid sequence of the sequence as shown in Fig. 8 (see Example 6).
  • the diagnostic agent may be used in the diagnosis of plasmodial infection, especially by parasites of the species P.
  • the diagnostic agent may be used to detect the presence of the plasmodial parasite or of a molecule related thereto in a sample as defined herein.
  • the diagnostic agent may be one which is suited for use in an agglutination assay in which the solid particles to which the antibody is coupled agglutinate in the presence of a polypeptide of the invention in the sample subjected to testing (see Example 7). In this type of testing, no labelling of the antibody is necessary. For most uses It is, however, preferred that the antibody is provided with a label for the detection of bound antibody or, alternatively (such as in a double antibody assay), a combination of labelled and unlabelled antibody may be employed.
  • the substance used as label may be selected from any substance which is in itself detectable or which may be reacted with another substance to produce a detectable product.
  • the label may be selected from radioactive isotopes, enzymes, chromophores, fluorescent or chemiluminescent substances, and complexing agents.
  • enzymes useful as labels are ⁇ -galactosidase, urease, glucose oxidase, carbonic anhydrase, peroxidases (e.g. horseradish peroxidase), phosphatases (e.g. alkaline or acid phosphatase), glucose-6-phosphate dehydrogenase and ribonuclease.
  • Enzymes are not in themselves detectable, but must be combined with a substrate to catalyze a reaction the end product of which is detectable.
  • a substrate may be added to the reaction mixture resulting in a coloured, fluorescent or chemiluminescent product or in a colour change or in a change in the intensity of the colour, fluorescence or chemiluminescence.
  • substrates which are useful in the present method as substrates for the enzymes mentioned above are H 2 O 2 , p-nitrophenylphosphate, lactose; urea, ⁇ -D-glucose, CO 2 , RNA, starch, or malate.
  • the substrate may be combined with, e.g. a chromophore which is either a donor or acceptor.
  • Fluorescent substances which may be used as labels for the detection of the components as used according to the of invention may be
  • Chromophores may be o-phenylenediamine or similar compounds. These substances may be detected by means of a spectrophotometer.
  • Radioactive isotopes may be any detectable and in a laboratory acceptable isotope, e.g. 125 I, 131 I, 3 H, 35 P, 35 S or 14 C.
  • the radioactivity may be measured in a 7-counter or a scintillation counter or by radioautography followed by densitometry.
  • Complexing agents may be Protein A, Protein G (which forms a complex with immunoglobulins), biotin (which forms a complex with avidin and streptavidin), and lectin (which forms a complex with carbohydrate determinants, e.g. receptors).
  • the complex is not in itself directly detectable, necessitating labelling of the substance with which the complexing agent forms a complex.
  • the marking may be performed with any of the labelling substances described above.
  • an antibody or a polypeptide of the invention may be coupled to a bridging molecule coupled to a solid support.
  • the bridging molecule which is designed to link the solid support and the antibody may be hydrazide, Protein A, glutaraldehyde, carbodiimide, or lysine.
  • the solid support employed is e.g. a polymer or it may be a matrix coated with a polymer.
  • the matrix may be of any suitable solid material, e.g. glass, paper or plastic.
  • the polymer may be a plastic, cellulose such as specially treated paper, nitrocellulose paper or cyanogenbromide-activated paper.
  • suitable plastics are latex, a polystyrene, polyvinylchloride, polyurethane, polyacrylamide, polyvinylacetate and any suitable copolymer thereof.
  • silicone polymers include siloxane.
  • the solid support may be in the form of a tray, a plate such as a mitrotiter plate, e.g. a thin layer or, preferably, strip, film, threads, solid particles such as beads, including Protein A-coated bacteria, or paper.
  • the polypeptide and antibody of the invention may be used in an assay for the identification and/or quantification of at least a form and/or a part of said polypeptide present in a sample.
  • the identification and/or quantification performed by the use according to the present invention may be any identification and/or quantification involving Plasmodium species molecules or a form of Plasmodium species molecules. Thus, both a qualitative and a quantitative determination of Plasmodium species molecules may be obtained according to the use of the present invention.
  • the identification and/or quantification may be performed for both a scientific, a clinical and an industrial purpose. As will be further described below, it is especially important in clinical routine to identify or quantify Plasmodium species molecules.
  • the sample may be a specimen obtained from a living organism such as a human or an animal, or an environmental specimen such as water.
  • the specimen may be blood, e.g. an erythrocyte enriched fraction, or a tissue sample e.g. comprising liver cells.
  • the specimen is urine.
  • the identification and/or quantification may serve the purpose of diagnosing an infection with a Plasmodium species in an organism, e.g. an animal or a human being.
  • the diagnosis is preferably
  • the identification and/or quantification may be performed by use of an assay in which the polypeptide or the antibody of the invention is employed.
  • the polypeptide or antibody may be part of an assay kit of a composition suitable for its intended use.
  • Such assay kits may comprise one or several layers and contain Plasmodium species molecules prepared by any of the methods described herein. This will be explained in further details below.
  • a drawback of some of the known methods for diagnosing malaria using clinical samples has been that known tests, when performed on samples of body fluids, principally whole blood, have not shown the specificity and sensitivity required for accurate diagnosis, and the one specific test, namely detection of the parasites in smears of peripheral blood obtained from an infected individual requires specially trained personnel, i.e. it cannot be performed as a routine analysis. Also it is unsuited as a screening analysis for the screening of a large number of patients.
  • the identification and/or quantification of Plasmodium species molecules in accordance with the present invention may be advantageous in accurate detection of e.g. recently acquired infection with Plasmodium species as readily available samples, in particular whole blood, plasma, serum or urine, may be used.
  • the malaria infection may be diagnosed by examining a sample, e.g. a blood or urine sample, for the presence of antibodies against
  • Plasmodium species molecules the presence of Plasmodium species molecules, and/or the presence of a DNA or RNA fragment encoding the Plasmodium species molecules. Also, the presence and amount of
  • Plasmodium species molecules in a vaccine may be determined in this manner.
  • one aspect of the Invention which Is contemplated to be novel and very interesting is the diagnosis of malaria infection performed on an urine sample.
  • the use of an urine sample in the diagnosis of malaria is an easy and convenient approach for the diagnosis as compared to the use of a blood or serum sample.
  • the antibody used in the method of the invention is a monoclonal antibody as this generally provides a higher precision and accuracy of the assay, at the same time possibly requiring less time to perform.
  • the monoclonal antibody may be obtained by the method described below. Antibodies possessing high avidity may be selected for catching techniques.
  • the antibody used in the present method is preferably in substantially pure form (purified according to suitable techniques or by the methods of the invention, see below) in order to improve the precision and/or accuracy of the assays of the invention.
  • the polypeptide or antibody of the invention is to be used for identification and/or quantification of Plasmodium species molecules it may be advantageous that the polypeptide or antibody is provided with a detectable marker or label.
  • the detectable marker may be any marker which may easily be identified by means of conventional techniques and equipment, such as a radioactively labelled marker, e.g. an isotope such as 125 I (G.Doring, H.J. Obernesser & K.
  • a complexing agent such as biotin may be a useful marker.
  • the determination of antibodies reactive with the polypeptide of the invention and being present in a sample may be carried out by use of a method comprising contacting the sample with the polypeptide of the invention and detecting the presence of bound antibody resulting from said contacting and correlating the result with a reference value.
  • the method of the invention employs some of the well known ELISA principles, e.g. direct (see Example 4), catching (see Example 5), competitive (see Example 6) and double enzyme linked immunosorbent.
  • a purified polypeptide preparation of the invention is attached to a solid support (e.g. a polystyrene microtitre tray (Nunc); the test solution to be measured is mixed with specific reference antibodies, e.g. the antibodies of the present invention, and this mixture is incubated with the solid support provided with the polypeptide preparation as mentioned above. After sufficient washing, enzyme-labelled anti-IgG-antibodies are added, and finally enzyme substrate is applied, see Example 6.
  • the ELISA and RIA methods are well established and may be carried out with existing laboratory equipment and may also be subjected to automation.
  • the methods of the inventions therefore has wide applicability in clinical laboratories for diagnostic purposes and for monitoring the results of vaccination procedures, and in the
  • Plasmodium species molecules or Plasmodium species molecules-like material may be determined both negatively and positively.
  • the method of the invention may be used for both
  • the polypeptide of the invention When the polypeptide of the invention is to be employed in an assay for determining the presence of Plasmodium species molecules in a sample, it may be in the form of a diagnostic reagent or a diagnostic agent. As will be apparent to a person skilled in the art several techniques may be applied in connection with such diagnostic
  • any part of said polypeptide is coupled to a solid support
  • an antibody against the component may then be added (see Examples 4 and 6).
  • the antibody is coupled to a solid support (see Examples 5 and 6).
  • any Plasmodium species molecules present in the sample is coupled to a solid support. It may then be incubated with the polypeptide component by addition of the component to the solid support followed by adding an antibody labelled with a
  • infection by Plasmodium species molecules in an organism or the presence of such in a sample may be detected by determining the presence of a DNA sequence related to the
  • Plasmodium species molecules using a DNA sequence of the invention The detection is based on homology between DNA sequences in the sample and the DNA sequence of the invention and may be performed by use of a diagnostic agent comprising a labelled DNA sequence
  • the DNA sequence may be labelled with any suitable label, e.g. selected from radioactive isotopes, enzymes, chemical modifying agents such as sulphonyl-introducing compounds and complexing agents such as biotin.
  • a nucleic acid molecule for the detection of the presence of DNA sequences related to Plasmodium species molecules in a sample may advantageously be carried out utilizing the principles of the polymerase chain reaction as described by Randall et al., Science, 1985, 230: 1350-1354, Randall et al., Science, 1988, 239: 487-491, and Stoflet et al., Science, 1988, 239: 491-494.
  • the polymerase chain reaction is a procedure used for the amplification of DNA present in a sample (see Examples 7 and 9). The procedure involves the use of two oligonucleotide primers which flank the nucleic acid molecule to be amplified.
  • the oligonucleotide primers may e.g. be 10- to 45-mers or more and comprise the flanking regions of glurp or be part of glurp .
  • the oligonucleotide primers are constructed so as to enable hybridization of one primer to the plus strand 5 'of the target DNA, and of another primer to the minus strand 5 'of the target DNA.
  • the preferred distance between the two primers is 100-2000 basepairs or more for diagnostic purposes, whereas longer distances could be accepted for preparative purposes.
  • the primers are hybridized with the opposite DNA strands to be amplified and are extended by using
  • DNA polymerase e.g. the Klenow fragment of E. coli DNA polymerase I or another useful DNA polymerase such as the Taq DNA polymerase, so as to synthesize a DNA sequence which is complementary to the DNA sequence to which the primers are annealed.
  • the DNA synthesized is denatured, e.g. by heating, from the "parent DNA strings” , and the parent strings as well as the newly synthesized DNA strings are subjected to a new PCR amplification cycle. In this manner, it is possible to obtain a substantial amplification of specific DNA sequences which are present in a sample.
  • FIG. 1 Crossed immunoelectrophoresis analyses of the specificity of the anti-fusion protein antibodies.
  • a volume of 20 ⁇ l of affinity purified antigen from in vitro P . falciparum culture supernatant was separated in the first dimension and run against 400 ⁇ l of immune serum in the second dimension.
  • B is with affinity purified immune serum in the intermediate gel.
  • precipitate representing GLURP is indicated by an arrow and the name; the double arrows indicate antigen 1.
  • GLURP is not precipitated on plate A, probably due to a low content of anti-GLURP antibodies in the serum used in the second dimension.
  • the localization of the precipitate representing GLURP on plate B indicates that the titer of anti-GLURP antibodies in the eluate is very high.
  • the two dimensions of the crossed immunoelectrophoresis is shown by the arrows l.D and 2.D.
  • Fig. 2 is a map of the plasmid pRD15 constructed of the plasmid pEX2 containing the ⁇ 15 insert in its EcoRI restriction enzyme cleavage site. Unique restriction enzyme cleavage sites and the most important non-unique restriction enzyme cleavage sites are indicated (BamHI, Bgll, EcoRI, Kpnl, Pvul, Pstl, Sail, Smal).
  • Pr is the rightward promoter of the ⁇ -phage, the arrow indicates the direction of transcription of this promoter.
  • Cro ' is the 5'-part of the ⁇ -phage cro-gene
  • lacZ' Is the 5 '-part of lacZ-gene encoding the N-terminal part of the ⁇ -galactosidase enzyme
  • Ori is the MBl-origin of replication, the arrow indicates the direction of replication,
  • bla is the gene encoding the enzyme ⁇ -lactamase conferring ampicillin resistance to the host organism.
  • the wavy line symbolizes the Cro'-gene and the lacZ ' -gene
  • the plasmid pRD15 encodes a fusion protein consisting of the N- terminal part of the Cro-protein, the N-terminal part of the ⁇ - galactosidase enzyme and GLURP.
  • the size of the plasmid pRD15 is 8857 basepairs.
  • Fig. 3 illustrates the hydropathy of the GLURP protein as determined by use of the indexes of Kyte and Doolittle.
  • Fig. 4 shows net charge of the GLURP protein, estimated for segments of 50 amino acids.
  • Fig. 5 shows the antigenicity according to Hopp and Woods of the GLURP protein.
  • Fig. 6 shows a dot diagram representing the optical densities measured by the antibody detecting ELISA for serum samples diluted 1:200 from malaria-immune patients as well as patients having diagnosed toxoplasmosis and schizostomiasis and serum from a control group, as described in Example 3.
  • Fig. 7 illustrates the nucleotide sequence of the DNA sequence glurp encoding GLURP.
  • the nucleotide sequence has been determined as described in Example 1.
  • the arrow indicates the stopcodon.
  • Fig. 8 illustrates the amino acid sequence deduced from the nucleotide sequence encoding the GLURP protein.
  • Fig. 9 shows a homology matrix with the sequence of the gene encoding GLURP represented along the X- and Y-axis.
  • a block of the first 30 bases from the X-axis is compared to the first 30 bases on the Y-axis and a dot printed at positions where at least 24 of the bases are identical (80% homology).
  • the block is then moved one base on the Y- axis to bases 1-31 and the comparison repeated and so forth to the end of the sequence.
  • the procedure is repeated with bases 2-32 from the X-axis and so on until the whole sequence has been compared.
  • Fig. 10 illustrates the CG content of the DNA sequence of glurp .
  • Fig. 11 shows the lysates of the ⁇ -gtll lysogen (lane 1) and the ⁇ 15 lysogen (lane 2), separated on a 7.5% SDS-PAGE.
  • the proteins were visualized by staining with Coomassie brilliant blue and the same proteins were blotted to nitrocellulose membranes (lanes 3 and 5: ⁇ gtll lysogen and lanes 4 and 6: ⁇ 15 lysogen).
  • Lanes 3 and 4 show the reaction of the antibodies affinity purified with antigen 1 as the ligand and lanes 5 and 6 show the reaction of a pool of sera from Danish donors.
  • Molecular weight markers used were ferritin (not reduced, not boiled) 440k, 220k; myosin 200k, ⁇ -galactosidase 116k; phosphorylase B 92.5k; bovine serum albumin 66k. (All k indicate kD).
  • Fig. 12 shows a Southern Blot of geographically different isolates of P . falciparum digested with Bell as explained in Example 1. ⁇ -DNA digested with Hindlll was used as molecular size markers.
  • Genomic DNA was digested with Bell, fragments separated by electrophoresis on a 1% agarose gel and blotted to nitrocellulose (Schleicher & Schuell) and probed with a nick-translated (nick-translation kit, Amersham) ⁇ 15-insert, labelled with the ⁇ -32p-dATP. The last washing was performed at 0.1 x SSC (Maniatis p. 447), 65°C, 10 minutes.
  • Fig. 13 A, B, C, and D Illustrate chromatograms obtained by
  • Fig. 14 illustrates a 7.5% SDS-PAGE run under reducing conditions:
  • Pharmacia Ferritin molecular weight marker Molecular weights in kD corresponding to the arrows (from top to bottom): 440, 220, 200, 116, 92, 66, 45. Lane 4, 5, and 9: Partially purified inclusion bodies from pRD15 (20, 30, and 10 ⁇ l, respectively). An extreme load of proteins was observed in the range from the top of the separationgel to the 45 kD marker.
  • Lane 6, 7, and 8 Inclusion bodies after 2700 mm gel filtration over a S400HR-column, volume applied to gel 10, 5 and 20 ⁇ l, respectively. The effect of the gel filtration chromatography was obvious. One band of relative molecular weight 300 kD was seen. The application of 20 ⁇ l of fusion protein in lane 8 resulted in overloading and the production of a smear. This figure combined with Fig. 13B, C, and D demonstrates the substantial purity of the protein.
  • Fig. 15 is a semilogarithmic plot of inhibition of the reactivity of human immune serum with native GLURP as a function of the dilution of GLURP used for absorption ( ⁇ ), GLURP could inhibit 92% of the reactivity which indicates that the recombinant protein is almost identical to the native protein.
  • GLURP could inhibit 92% of the reactivity which indicates that the recombinant protein is almost identical to the native protein.
  • similar amounts of ⁇ - galactosidase were used instead of GLURP for the similar procedures (x). No inhibition was observed. Dilutions were made from a 1 mg GLURP / ml solution in PBS.
  • Fig. 16 is a semi-logarithmic plot showing the GLURP detecting ELISA employed on supernatant from in vitro P . falciparum culture
  • the dilution of the specimens was 1:100, capturing antibody was purified rabbit-anti-fusion protein antibodies diluted 1:320 from a 4 mg/ml stock. Detecting antibody was mouse-anti-fusion protein antibodies diluted 1:500 from serum. The conjugated rabbit- anti-mouse immunoglobulin (DAKOPATTS P260) was diluted 1:1000.
  • Fig. 17 Epitope mapping. Partly purified protein, containing the major repeat area of GLURP was tested for T-cell epitopes.
  • the figure shows the proliferative response of T-cells from (A) 16 malaria-immune donors from the Gambia, and (B) 8 non- immune European donors.
  • T-cells from 13 out of 16 malaria- immune donors responded with a proliferative index above 2.5, while only one of the non- immune donors, a malaria convalescent, responded significantly,
  • the stimulation index was calculated by dividing the geometric mean of a triplicate of measurements with the geometric mean of three control measurements.
  • Fig. 18 is a semi-logarithmic plot showing the results obtained in an antibody detecting ELISA analysis of antibodies present in the sera of rabbits which had been immunized with the fusion protein as described in Example 12A.
  • Fig. 19 is a semi-logarithmic plot showing a competition assay:
  • Rabbit anti-fusion protein antibodies competed with human malariaimmune serum for the fusion protein coating.
  • the rabbit antibodies were able to extinct the reactivity of the human antibodies as described in Example 12A.
  • Fig. 20 A, B, C, and D Titrations of monkey serum obtained from the four monkeys immunized with the fusion protein.
  • the sera were collected on day -132 ( ⁇ ), day 0 (x), day 14 (o), and day 28 (X), with respect to the day of immunization.
  • Analyses were performed with the antibody detecting ELISA described in Example 3 coated with 0.125 ⁇ g of fusion protein/well.
  • the binding of monkey anti-fusion protein antibodies was detected by rabbit-anti-monkey serum antibodies diluted 1:1000, the binding of rabbit antibodies was subsequently detected by porclne-anti-rabbit antibodies diluted 1:1000.
  • A represents the titration curves for monkey number 864.
  • B represents the titration curves for monkey number 865.
  • C represents the titration curves for monkey number 866.
  • D represents the titration curves for monkey number 867.
  • the malaria isolate used for constructing the genomic library was the Kenyan isolate, F32.
  • the isolates used for the Southern Blotting were from Africa (F32, D25), Burma (D51), Senegal (D28), India (D41), Liberia (L1), Kenya or Kenya (D50) and Kenya (K1). All the isolates are from patients who had travelled in the country indicated as the origin. Isolate D50 is from a patient who had travelled in Kenya and in Africa. All malaria isolates are available at Statens Seruminstitut, Copenhagen, Denmark. Human sera used for the screening of the genomic library and for the characterization of proteins were from Africa and Indonesia. Antibodies predominantly recognizing antigen 1 are purified from Africa and Indonesia.
  • African malaria-immune sera using a chromatographic affinity purification with antigen 1 as the ligand were used: M13, pUC9, ⁇ gt11, pEX2.
  • Microtiter ELISA plates no 4-39454 obtained from NUNC.
  • ELISA reader Immunoreader, NJ2000, TECHNUNC.
  • Immunowasher 12 obtained fromN NUNC. Buffers :
  • Carbonate buffer pH 9.6 (1.59 g Na 2 CO 3 , 2.93 g NaHCO 3 to 1000 ml with distilled water).
  • Washing buffer (29.2 g NaCl, 0.2 g KCl, 0.2 g KH 2 PO 4 , 1.15 g
  • Dilution buffer pH 7.2 (10 g Bovine albumin, 2 ml phenol red 0.5%, to 1000 ml with washing buffer, pH adjusted to 7.2 with sodium hydroxide).
  • Colouring buffer pH 5.0 (7.3 g citric acid ⁇ H 2 O, 11.86 g Na 2 HPO 4 ⁇ 2 H 2 O, to 1000 ml with distilled water).
  • Colouri-ng substrate solution 40 mg OPD is dissolved in 100 ml colouring buffer supplemented with 40 ⁇ l of hydrogen peroxide). The container for this solution was wrapped in tin foil to avoid exposure to light. The solution could be used for 1 to 2 days stored at 4°C.
  • UV-monitor Pharmacia Fine Chemicals, Uppsala, Sweden, model UV-1, 280 nm.
  • Concentration cell Amicon cell, model 202, ultrafliter PM 10
  • Fraction collector Redirac , LKB , Sweden.
  • IgG Intragen 1 IgG was isolated from EDTA-plasma of a known malaria-immune African adult by salting out and ion exchange as described in details in the literature, e.g. Harboe, N and Ingild, A: Immunization, isolation of immunoglobulins and estimation of antibody titer. Scand. J. Immunol. 2, suppl. 1:161,1973. By crossed immunoelectrophoresis (CIE), the plasma was shown to contain antibodies against antigen 1 and
  • Antigen 2 as described in (1).
  • the crossed immunoelectrophoresis was carried out essentially as described by Jepsen, S and Axelsen, NH in (1).
  • 140 mg of the IgG antibodies was coupled to 15g CNBr activated Sepharose 4B from Pharmacia Fine Chemicals. The procedures of the manufacturer were followed.
  • Antigen from the supernatant of malaria culture (P . falciparum, F32) was purified as described by Jepsen, S and Andersen, B J in (2).
  • a pool of antigen purified on the immune IgG column was coupled to CNBr activated Sepharose 4B according to the manufacturers instructions. It was observed fortuitously by analyzing the passage of this coupling by crossed immunoelectrophoresis as described above that it contained antigen 1 exclusively, being due, either to an excessive amount of antigen applied to the used mass of CNBr Sepharose, and/or being due to a lower efficiency of the coupling of antigen 1 than of the other antigens in the pool to CNBr activated sepharose. The run- through was concentrated and coupled to 2.3 g (dry weight) of CNBr Sepharose 4B following the procedure of the manufacturer.
  • SDS-PAGE Sodium Dodecyl Sulphate Poly aery lamide Gel Electrophoresis Analysis of proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with the BioRad Protean 2 or Mini-Protean 2 system using a discontinuous gel system with a 3% stacking gel and a 7.5% separating gel. All reagents for gel electrophoresis were obtained from BioRad.
  • a volume of 50 ⁇ l of each sample to analyse was mixed with 1/3 volume loading buffer (4% Sodium dodecyl sulfate (SDS), 0.4 M dithiotreitol, 0.08 M Tris-HCl pH 7.8, 10% glycerol), boiled for 5 minutes, and separated by electrophoresis in the above mentioned gel system (reference:
  • the proteins were initially separated on SDS-PAGE as described above and then transferred electrophoretically from the gel to a 0.22 ⁇ m nitrocellulose filter (Schleicher und Schuell). Blotting was done at a field strength of 6 V/cm for 5 hours at 4°C with the gel and membrane submerged in transfer buffer. After blotting, the nitrocellulose filter was blocked with a washing buffer containing 0.5% Tween-20 solution and Blots were incubated for one hours with sera or purified antibody against antigen 1 diluted appropriately in washing buffer. After extensive washing (3
  • CIE was performed on glass plates 7 x 5 cm in 1% agarose gel (Litex, Glostrup, Denmark, type HSA) in Tris-barbitol buffer pH 8.6 ionic strength 0.02 by running 20 ⁇ l of the affinity purified soluble antigens in the first dimension gel at 10-15 Volt/cm until a parallel blue albumin marker had migrated 2,6 cm.
  • the second dimension electrophoresis was run perpendicular to the first dimension gel at 2 Volt/cm for 18 hours into a gel containing 12 ⁇ l/cm 2 human Liberian immune serum as defined above. The plates were washed and pressed three times and stained with Coomassie brilliant blue R250.
  • Parasite cultures were centrifuged at 2400 x g for 5 min. Cell pellets were washed in 5 volumes sterile 0.9% NaCl, centrifuged at 2400 x g for 5 min and incubated in 5 volumes of a 0.01% saponin solution in sterile water for 10 min at room temperature. After centrifugation at 4500 x g for 10 min, the saponin treatment was repeated and the pellets were finally washed in 5 volumes of sterile isotonic NaCl and centrifuged at 800 x g for 10 min. The cell pellet was discarded and the supernatant was used for SDS-PAGE.
  • Heparinised venous blood was collected from Gambian donors.
  • Non- immune (control) samples were obtained from from from Europeans expected not to be malaria-immune.
  • Lymphocyte proliferation assays were performed as described previously (Riley, EM et al., 1988). Briefly, mononuclear cells (MNC) were separated by density centrifugation and stimulated with Purified Protein Derivative of tuberculin (PPD) or with Phytohemagglutinin (PHA) or control buffer containing ⁇ -galactosidase. Assays were performed in triplicate in roundbottomed microtiter plates and cultures were incubated for either 3 days (PHA) or 7 days (PPD and antigens) at 37oC in 5% CO 2 . Proliferation was determined by
  • LB medium 10 g NZ amin, 5 g yeast extract (Difco), 5 g sodium chloride, 2 g magnesium sulphate 7 H 2 O, adjusted to pH 7.5, distilled water to 1 litre.
  • the components are autoclaved at 120°C for 1/2 hour and then stored in sterile bottles at 4oC.
  • LB plates with agar 10 g NZ amin, 5 g yeast extract (Difco), 5 g sodium chloride, 15 g agar (Difco), volume adjusted to 1000 ml with distilled H 2 O and the total composition autoclaved at 120°C for 1/2 hour and distributed in Petri-dishes, the plates optionally being supplemented with 50 mg/l ampicillin (Ampicillin obtained from DAK).
  • LB top agarose used for ⁇ gtll in Y1090 5 g NZ amin, 2.5 g yeast extract (Difco), 2.5 g sodium chloride adjusted with distilled water to 500 ml, 0.35 g agarose In each 100 ml bottle. 50 ml of the above solution is poured into each 100 ml bottle and all bottles autoclaved at 120°C for 20 minutes.
  • Electrode buffer 0.025 M Tris.glycine pH 8.3, 0.1% SDS.
  • Transfer buffer 0.25 M Tris.glycine pH 8.3, 20% methanol.
  • the red blood cells were sedimented by gravity.
  • the suspension was centrifuged at 2000g av for 5 minutes and the supernatant was removed.
  • the pellet was resuspended in 0.01% saponin in isotonic saline and incubated for 10 minutes at room temperature.
  • the pellet was resuspended in a volume of 0.01% saponin in
  • isotonic saline constituting approx. 5 times the volume of the pellet and incubated at room temperature for 5 minutes followed by centrifugation at 3000g av for 10 minutes.
  • the pellet was washed in a volume of isotonic saline constituting approx. 5 times the volume of the pellet and centrifuged at 3000g av for 10 min.
  • the pellet was suspended in a volume of DNA buffer (100 mM Tris- HCl pH 8.0, 1% SDS, 50 mM EDTA, 0.2 M NaCl) constituting approx. 5 times the volume of the pellet.
  • DNA buffer 100 mM Tris- HCl pH 8.0, 1% SDS, 50 mM EDTA, 0.2 M NaCl
  • RNase A which had been boiled for 10 minutes was added to 50 microgram/ml and the suspension was incubated at 37°C for 1 hour.
  • Proteinase K was added to 100 microgram/ml, and the suspension was incubated at 50°C for 1 hour.
  • the pellet was redissolved in a suitable volume of 10mM Tris-HCl pH 7.5, ImM EDTA.
  • the concentration of DNA was estimated by measurement of OD 260 and OD 280 according to Maniatis et al., op cit., p. 468.
  • DNA fragments were madeblunt endedby filling outwith T4 DNA polymerase in the presence of the four types of deoxynucleotide, as described in Maniatis op . cit. , p. 117-121.
  • EcoRI linkers from New England Biolabs were ligated to the DNA fragments as described in Maniatis op . cit. , p. 243-246.
  • the resulting DNA fragments with EcoRI linkers were digested with EcoRI (Boehringer-Mannheim) and ligated to lambda gtll arms (Promega Biolabs). The ligations were performed with T4 DNA ligase (Amersham).
  • the recombinant lambda genomes were packaged with a packaging mix (Promega biolabs) according to the instructions of the manufacturer.
  • the library was then used to infect Y1090 and plated on LB medium with agar.
  • Digestions of the insert was performed with exonuclease III (the Erase-a-base system from Promega, USA) in order to produce fragments of a length suitable for the sequencing of the 3' region of the gene being a highly repetitive region. The procedure of the manufacturer was followed.
  • the plaque estimated to be of value was picked with a Pasteur pipette, put into SM-medium (Maniatis et al., op cit., p 70) and shaken at room temperature for 2 hours after which the phages were liberated to the medium.
  • SM-medium Maniatis et al., op cit., p 70
  • the phage clone (termed ⁇ 15) selected for lysogenization was initially amplified as described by Huynh H et al., Constructing and screening cDNA libraries in lambda gt10 and lambda gtll, in DNA cloning - a practical approach., IRL Press 1985.
  • the amplified phagestock having a titer of >10 10 plaque forming units/ml, was used for infection of E.coli Y1089 to establish the ⁇ 15 as a lysogen in the bacteria.
  • the colonies on the plates were tested for temperature sensitivity by spreading bacteria from each colony on identically marked places of the two LB plates, and subsequently placing the plates at 30°C and 42°C, respectively. Colonies growing at 30°C but not at 42°C are assumed to be lysogens.
  • IPTG Isopropyl ⁇ -D-thlogalactopyranoside obtained from Sigma Biochemical Company
  • the culture was centrifuged at 27°C at 2000 g av for 10 minutes, the pellet resuspended in PBS (1/10 the volume of the culture), sonicated 3x20 seconds at maximum output ( 150 watt MSE Ultrasonic disintegrator) and frozen at -80°C.
  • Analysis of the lysates by sodium dodecyl sulphate polyacrylamide gel eletrophoresis (SDS-PAGE) and Western blotting was performed with the BioRad Protean 2 system as described in MATERIALS AND METHODS, with sera from non-immune donors diluted 1:800 and affinity purified antibodies against antigen 1 diluted 1:200, cf. Fig. 11.
  • DNA from the ⁇ 15 lysogen Y1090 E . coli was prepared according to standard procedures described in Maniatis et al., (pp. 76-94). To excise the ⁇ 15 insert originating from Plasmodium falciparum, the DNA was digested with the restriction enzyme EcoRI from Amersham using 10 units per ⁇ g DNA for one hour at 37°C. A buffer consisting of 100 mM Tris-HCl, pH 7.5, 50 mM sodium chloride and 10 mM magnesium chloride was used. The expression vector pEX2 described in EMBO. J. vol. 3, 1984, pp. 1429-1434, C. S. Stanley and J. P.
  • Excised ⁇ 15 inserts and dephosphorylated linearised pEX2 vectors were mixed and ligated at 4°C oveimight with T-4 DNA ligase from Amersham. Essential features of this construction is illustrated in Fig. 2.
  • POP 2136 is a ⁇ phage derivative which has been constructed in the following way: first the 2.3 kb Bglll fragment of ⁇ phage, carrying the CI857 allele and the P R promoter, was cloned into the BamHI site of a polylinker as shown below:
  • POP2136 orientation malT, P R , C1857, malPQ
  • the resulting POP2136 is Mal- at both temperatures, 30°C and 40°C, and is ⁇ immune.
  • Buffer used for Seal 6 mM Tris- HCl, 150 mM sodium chloride, 6 mM magnesium chloride, 6 mM 2-mercaptoethanol bovine serum albumin, 100 ⁇ g/ml, pH 7.5, at 37 °C.
  • Buffer used for BamHI 10 mM Tris-HCl, pH 8.0, 7 mM magnesium chloride, 100 mM sodium chloride, 2 mM 2-mercaptoethanol, 0.01% bovine serum albumin at 37°C. The digests were analysed on a 1% agarose gel, separation time 4 hours, 100 Volts.
  • Clones that gave rise to an essentially linearised recombinant pEX2 (the distance between the Seal and the BamHI sites are a few hundreds of bases) were chosen for further characterization.
  • This clone has been deposited with Deutsche Sammlung von Mikroorganismen, DSM, on 15 September 1988 under Accession No. 4815.
  • the clone selected for further expression (DSM 4815) was maintained on LB plates supplemented with ampicillin and stored at 4°C. Longer term storage was secured by -80°C storage of an overnight culture supplemented with glycerol to 20%. An overnight culture grown at 28 ° C and supplemented with ampicillin to 50 mg/l was diluted 100 - fold in LB medium supplemented with ampicillin to 50 mg/l. Recombinant bacteria were grown in Pregl flasks on an orbital shaker at 28 °C until the OD 600 reached 1 .0. The temperature was increased to 42°C for 1 hour and cultivation continued until the OD 600 reached
  • the E. coli cells were then harvested by centrifugation in a Sorval superspeed centrifuge using the GS-3 head at 5000 r.p.m. (2000 g av ) for 10 minutes at room temperature.
  • the pellet was then resuspended in TEN (50 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 150 mM sodium chloride).
  • the pellet was resuspended in 1/100 the volume of the original culture of TEN, and the cells were disintegrated in a French press from American Instruments Company, Maryland, USA, at a pressure of 18000 psi.
  • the material was centrifuged at 4000 g av at 4°C for 10 minutes and the supernatant discarded.
  • the pellet was washed in a volume of TEN equivalent to 1/100 of the original culture volume.
  • the suspension was again centrifuged for 1000 g av for 10 minutes at 4°C. This procedure was performed two more times.
  • the resulting pellet consisting of inclusion bodies was resuspended in 1/100 the volume of the original culture of denaturing buffer consisting of 5 M
  • guanidinium-HCl 50 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol, 1 mM EDTA. The material was left overnight to be gently shaken at 4°C. Soluble material was separated from insoluble material by
  • the urea concentration was then lowered gradually to zero over a period of approx. 10 hours, maintaining the DTT concentration at 1 mM.
  • the DTT concentration was lowered to zero, the dialysis being against, e.g., 20 mM Tris HCl pH 7.5 at room temperature for approx. 2 hours.
  • the purpose of this procedure was to keep the fusion protein solubilized. The procedure allowed 1) the protein to fold to Its normal structure and 2) to regain its internal disulphide bridges. In case a too high rate of precipitation is observed, the procedure is repeated with the protein dissolved in 5 M urea, 20 mM Tris HCl pH 7.5 and 1 mM DTT.
  • Fractions predominantly containing fusion protein have been pooled and concentrated.
  • the concentration procedure was performed by establishing an osmotic gradiant between the fusion protein containing solution inside a bag of dialyses membrane and PEG20000 flakes deposited on the outside of the bag. Using this method the fusion protein remained in a buffer consisting of
  • guanidinium hydrochloride, DTT and pH maintaining buffer of the required concentrations The volume of the pool fraction was thus reduced to a volume optimal for application on the gel filtration column, e.g. 10-15 ml. This procedure has been repeated one more time resulting in a total separation of the fusion protein and the E.
  • fusion protein refolded as outlined above
  • a column with mouse monoclonal anti- ⁇ -galactosidase antibodies coupled to CNBr sepharose 4B, obtained from Pharmacia is used.
  • the antibodies used are purified from hybridoma supernatants using a protein A and/or a fusion protein column and are coupled to the cyanogen bromide activated Sepharose according to the procedures of the manufacturer. Pooled fractions from gel filtration shown to contain the major part of the fusion protein are applied to the column; the amount of fusion protein not exceeding the total binding capacity of the column. This is monitored by examining the passage with the ELISA utilizing the anti-y9-galactosidase antibodies.
  • the column is then washed with 3 bed volumes of column buffer and eluted using 3 M potassium thiocyanate (KSCN) in column buffer without NaCl.
  • KSCN potassium thiocyanate
  • the collected fractions are dialyzed against PBS and analyzed using the ELISA for detection of ⁇ -galactosidase. Concentration to approx. 2 mg/ml is performed in an Amicon diaflow concentrator cell.
  • the material is passed through a polymyxin B column and checked for endotoxin activity by use of the limulus amoebocyt lysate assay.
  • the competition assay was performed in two phases: 1) an absorption phase where antibodies from human malaria-immune serum reacted in an ELISA well with a coating of the fusion protein,
  • the other control was coating of the first phase absorption ELISA wells with ⁇ -galactosidase obtained from Boehringer-Mannheim.
  • ⁇ - galactosidase is the non-malarial part of the fusion protein. This control was included to rule out the possibility of unspecific interaction of antibodies and the non-malaria part of the fusion protein or the possibility that the absorption was due to a nonspecific interaction of any protein in an ELISA well and the antibodies in the serum.
  • Percent inhibition excerted by a given amount of fusion protein was calculated by subtracting the OD obtained on a coating of culture medium presented on rabbit-anti-GLURP antibodies from the OD obtained on a coating of culture supernatant presented on the same antibodies and dividing by the difference between the maximum OD value obtained with an unabsorbed human-immune serum of the above mentioned dilution reacting with a coating of culture supernatant presented on the above mentioned antibodies and the OD value obtained by the reacting of the unabsorbed human-immune serum with a coating of culture medium presented on rabbit-anti-GLURP antibodies. That is, percent inhibition (P) by given absorption, (Z) is
  • the plates were left overnight at 4°C in a humid chamber. (Coating of the ELISA plates can alternatively be performed by incubation for 2 hours in the humid chamber at room temperature on an orbital shaker.) Washing was performed with a Technunc Immunowasher 12 using the washing buffer. Each well was flooded with buffer 4 times. After washing, the plate was emptied and 100 ⁇ l of a conservation buffer applied to each well.
  • a rabbit anti-human-IgG conjugated to horseradish peroxidase (Dakopatts P214 dilution 1:10,000) was then applied to the well in a volume of 100 ⁇ l. (For detection of antibodies in species other than the human, the horseradish peroxidase conjugated antibody is directed against the IgG of the species in question.)
  • the plate was incubated at room temperature for one hour on an orbital shaker, then washed and emptied as described above. 100 ⁇ l of colouring buffer was applied to each well and the plate incubated for 1 minute. The colouring buffer was removed from the wells and 100 ⁇ l of colouring substrate applied to each well followed by incubation for 20-30 minutes.
  • the colouring process was stopped by applying 150 ⁇ l IM sulfurie acid to each well.
  • the optical density at 490 nanometers was measured on a Titertek ELISA reader and the results are illustrated in Fig. 6.
  • the ratio between positive and negative readings was about 15, which is very satisfactory.
  • the average of the 93 Danish donors was approx. 0.067 absorbance units with a standard deviation of 0.057.
  • a typical value for a malariaimmune patient was above 1.5.
  • the negative results obtained from the African donors were verified by analysis of the sera by use of crossed immune-electrophoresis.
  • the group of Liberian donors giving values below the 95% did not have any precipitates interpreted as GLURP in CIE.
  • These patients probably have not had malaria due to pharmaceutical prophylaxis or a place of living with no malaria transmission, i.e. in the capital of Liberia.
  • the detection of antibodies against the fusion protein sometimes has to be carried out in species against the antibodies of which one does not possess antibodies. This, for example, was the case in the immunization experiment carried out in the monkeys mentioned in Example 12C. This obstacle was initially circumvented using a competitive ELISA principle. Later on, rabbit-anti-monkey serum antibodies were available. In this assay, ELISA plates coated with fusion protein in usual amounts were used. The monkey antibodies in varying dilutions from 1:5-1:640 were put into the wells of the above mentioned ELISA plates in a volume of 50 ⁇ l per well. The monkey antibodies were incubated alone in the wells for 15-30 minutes after which period of time 50 ⁇ l of a human immune serum was applied.
  • human immune serum The dilution of human immune serum was chosen so as to put the human antibody on the linear part of the titration curve in order to obtain the maximum variation in the amount of antibody bound to the fusion protein coating. Incubation was prolonged 1 hour after the addition of the human serum. Binding of human immunoglobulins was detected by rabbit anti-human immunoglobulin antibodies (P214) diluted 1:10000. Visualization was performed as above.
  • Measurements of GLURP in serum or secretions from human or animal bodies, especially in urine, can be performed using the ELISA technique or using the principle of competitive ELISA.
  • Rabbit anti-fusion protein antibodies were purified from rabbits immunized as described in Example 12A. The purification of the antibodies were according to the procedure of Harboe, N. and Ingild, A.: Immunization, isolation of immunoglobulins and
  • the antigen was incubated in the well for at least two hours, preferably over night.
  • the detection of binding of GLURP to the rabbit antibodies coated to the bottom of the well was performed either by the use of human anti-P. falciparum antibodies or by the use of mouse anti-fusion protein antibodies (see Example 12B).
  • Human antibodies were used in dilutions of 1:200 to 1:2000.
  • Antibodies applied for this purpose were incubated for one hour at room temperature on a shaking table.
  • the dilution of the mouse anti-fusion protein was 1:500 to 1:1000.
  • the binding of human antibodies to GLURP was detected with rabbit anti- human IgG (DAKOPATTS P214) diluted 1:1000.
  • the binding of mouse antibodies was detected using a rabbit anti-mouse IgG conjugated to peroxidase (DAKOPATTS P260) diluted 1:1000.
  • the above described ELISA has been used for comparison of the contents of protein in various batches of the fusion protein with the contents of a ⁇ -galactosidase standard. This was done in order to estimate the contents of different batches.
  • the detecting antibody was mouse monoclonal anti- ⁇ -galactosidase
  • the sensitivity of the GLURP detecting ELISA used for detection of GLURP in blood, serum or urine may be further increased by the use of affinity purified antibodies both in the capturing and in the detection layers.
  • affinity purified antibodies both in the capturing and in the detection layers.
  • One or both of these layers may consist of
  • these layers may be composed of antibodies, enzymatically processed to Fab fragments.
  • the detecting antibody may be directly labelled to the detecting
  • Porcine anti-rabbit antibodies conjugated to peroxidase (DAKOPATTS P217) was used in a dilution 1:1000, incubated for 1 hour on a rocking platform at room temperature. The plate was washed as described above. 100 ⁇ l of colouring buffer was applied to each well for 1 minute, then removed and 100 ⁇ l of colouring substrate was applied to the well. The colouring reaction was allowed to continue for 20-30 minutes, and then stopped with 150 ⁇ l, IM sulfurie acid. The optical density at 490 nanometers was measured on an ELISA reader.
  • the main advantage of the competitive principle is the speed with which it can be performed. Generally it med that the detection limit for the presence of GLURP in culture supernatant was dilutions of 1:10-1:100. Generally it med that low concentrations of rabbit antibody to a certain extent increased the sensitivity as did low amounts of fusion protein in the solid phase.
  • An alternative design of the competitive ELISA is to coat the wells of the ELISA plate, as described above, with antibodies (obtained from any species or cell system by immunization with the fusion protein).
  • a concentration of antibodies of 0.004 ⁇ g/ml to 400 ⁇ g/ml in carbonate buffer is used.
  • 50 ⁇ l of the sample is mixed with 50 ⁇ l of a dilution of the fusion protein in dilution buffer.
  • a series of consecutive dilutions is used, the dilutions ranging from 1 mg/ml to 1 pg/ml in dilution buffer or another range of dilutions which is found appropriate considering the type of sample.
  • the visualization of fusion protein bound to the antibodies in the well may be performed using a fusion protein labelled as described herein with a fluorescent molecule or an enzyme capable of hydrolyzing a substrate and thereby changing the absorbance of the substrate at a given wavelength or any other principle.
  • an antibody directed against the ⁇ -galactosidase part of the fusion protein may be used to detect the presence of the fusion protein, either by using an anti- ⁇ -galactosidase antibody labelled itself or using another antibody with specificity against the anti- ⁇ -galactosidase antibody conjugated to any labelling principle in the next layer.
  • the detection of proteins specific for P. falciparum can be performed as described in Examples 5 and 6.
  • the detection can be simplified passing the urine sample through a membrane coated with antibodies against GLURP thereby capturing GLURP and subsequently detecting the presence of GLURP by an antibody conjugated to an agent for visualization, i.e. alkaline phosphatase using the appropriate substrate.
  • the detection of DNA specific for P. falciparum can be performed as described in Example 9.
  • Urine from individuals suffering from acute malaria has been collected, preserved with Sodium azide and frozen at -20oC before shipment to our laboratory.
  • An alternative design of an assay for the presence of GLURP in urine is to coat particles, e.g. latex particles, with antibodies against GLURP (obtained from any species or cell system by immunization with fusion protein). These particles are then suspended in a volume of the urine sample, if necessary in a buffer. The presence of GLURP in the urine will be detected as the occurrence of agglutination and precipitation of the particles.
  • particles e.g. latex particles
  • antibodies against GLURP obtained from any species or cell system by immunization with fusion protein
  • EXAMPLE 8 Determination of the Optimal Amount of Antigen Used for Coating of ELISA-plate.
  • An ELISA plate was coated with decreasing amounts of protein starting with 100 ng/well (corresponding to 0.5 ⁇ l/well) of an aqueous solution of the fusion protein with a concentration of 2 mg/ml .
  • the antigen was diluted in carbonate buffer, pH 9.6. Consecutive two-fold dilutions were applied to the ELISA plates. Five sera were used for this experiment.
  • An immune serum from Liberia a pool of immune sera previously giving a high OD, three sera from donors of which one was known to give a very low OD and two to give a medium OD reaction, although they had no known exposition for malaria antigens.
  • PCR polymerase chain reaction
  • amplification cycle used was 94°C for 1 minute to denature the DNA, 65°C for 2 minutes to anneal the primers and 72°C for 8 minutes to run the polymerase reaction, repeated 35 times.
  • the product of the amplification was checked by calculating the size of the DNA after agarose gel electrophoresis, the AD-product by calculating the size of fragments after restriction enzyme digestion of the amplification with Kpnl.
  • the amplificate can be further analysed by transferring the DNA to a nitrocellulose membrane as dot Blot or Southern Blot, and
  • a radioactively labelled probe can be used, a probe labelled with a fluorescent molecule or a probe coupled to an enzyme which is able to hydrolyse a substrate, the hydrolysis of which is revealed by an absorbance variation for a given wavelength of radiation, e.g. to horseradish peroxidase.
  • the visualization could be amplified using intermediate steps, e.g. involving a biotinylated probe reacting with avidin or streptavidin conjugated with an enzyme or a fluorescent molecule; alternatively a chemically modified probe reacting with an antibody directed against the chemical modification conjugated to an enzyme or fluorescent molecule.
  • the visualization is then performed with the appropriate system, i.e.peroxidase staining and absorbance measurement, measurement of light emission or the like.
  • peptides or proteins derived from the amino acid sequence of GLURP may be used for immunization purposes. These peptides or proteins could be fragments or rearrangements of the amino acid sequence produced in P. falciparum, alternatively produced in yeast, mammalian cell cultures or in any other organism, e.g. in E . coli as a fusion protein, in E.
  • coli or in any other organism as the gene product itself, e.g using the vector described by Nagai and Th ⁇ gersen in Methods of Enzymology, vol 153, chapter 29, Academic Press inc., 1987, or, alternatively, using the gene or components of the gene in any arrangement inserted in a vector, e.g. vaccinia, useful for gene transfer to the animal to be immunized and after transfer being able to express the gene product in a way that confers immunity to the animal against the malaria parasite.
  • a vector e.g. vaccinia
  • Gene fragments encoding immunologically important regions are sequenced to relate them to the nucleotide sequence and thereby to the amino acids sequence.
  • T-lymphocyte epitopes the gene encoding GLURP has been analyzed with the AMPHI program (Margalit et al. in J Immunol . 138 , pp. 2213-2239, 1987). According to this analysis, potential helper T cell epitopes were the following sequences (which were also shown above), amino acid residues in parentheses:
  • a total of 202 individuals living in a malaria endemic area in the Gambia were tested for induction of lymphocyte proliferation by the fusion protein.
  • the bacteria carrying pMBB98 was grown to an OD 600 of 0.5 in LB- medium, the expression induced by adding IPTG to a total concentration of 1 mM and grown over night.
  • the cells were then harvested by centrifugation and resuspended in 20 ml 10 mM Bis-Tris pH 6 and crushed by passing through a French press. Cell debris were removed by centrifugation 20.000 G for 1 hour and 8 ml of the supernatant was applied on a 100 ml DEAE fast flow sepharose matrix (Pharmacia-LKB, Sweden).
  • the column was subjected to increasing concentrations of NaCl in Bis-Tris pH 6 on an FPLC, and 5 ml
  • the ⁇ -galactosidase activity was used to select fractions to
  • fractions containing the fusion protein which was eluted at an NaCl concentration of approximately 0.6 M.
  • the selected fractions were pooled, dialysed against PBSA and concentrated to 10 ml by dialysing against solid PEG 20 000.
  • T-cells from malaria-immune donors The ability to induce a proliferative response in T-cells from malaria-immune donors was tested. T-cells from 13 out of 16 donors responded, whereas only T-cells from one out of 8 non-immune donors responded (Fig. 17). The responding non-immune donor was convalescent from a malaria attack 4 weeks earlier.
  • T-cell epitopes are located within or flanking the repeat area.
  • Two domains within the sequence were found by the AMPHI program to contain potential T-cell epitopes (aa 333-343, and aa 600-613, above). Further investigations of these epitopes using synthetic peptides are under preparation.
  • T-cells from the non-immune convalescent reacted indicates that a single malaria attack could be sufficient for boosting a response, and we propose the usage of such highly potent T-cell epitopes from GLURP as components of a subunit vaccine against malaria.
  • GLURP gene from different isolates are amplified using PCR, cloned into the pUC-vector and sequenced. The most conserved epitopes are produced as synthetic peptides and tested for T-lymphocyte proliferation stimulatory properties and/or lymfokine secretion.
  • B- and T-cell epitopes are then put together, either on the nucleotide level to be produced in an organism or put together as amino acids in synthetic peptides.
  • the combination of the epitopes should be tested for the preservation of the properties of the separate epitopes. That is, the immune stimulating properties of the separate epitopes could be abolished by putting them close together due to interaction between the amino acids.
  • the protein content of pooled fractions was determined according to the Bio-Rad method (Bradford, M M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254,1976) showing a concentration of 2 mg/ml. Approximately 75 mg of fusion protein were coupled to 15 g (dry weight) of CNBr activated
  • sepharose 4B obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. All procedures for coupling given by the manufacturer were followed. 3 ml of human malaria-immune serum from Liberia was diluted 1:10 in column buffer. The material was filtered and then applied to the column using a pump, flow rate 20 ml/hour. Washing was performed with 3 bed volumes of the column buffer. Elution was done with 3 M potassium thiocyanate in column buffer without NaCl. Fractions eluted from the column showing UV absorption above that of the elution buffer were pooled and dialyzed against the column buffer without NaCl. Concentrated on an Amicon diaflow concentration cell to 1/3 the original volume of the serum.
  • an ELISA coated with the fusion protein for the monitoring of the run-through may be used.
  • the run-through should not contain major amounts of antibodies against the fusion protein.
  • Three rabbits were immunized with a vaccine composed of 1 volume of 2 mg/ml fusion protein in PBS produced as described in Example 2 and 1 volume of Freunds incomplete adjuvant in a volume ratio of 1:1. Each rabbit received 0.1 ml subcutaneously three times with an interval of one week. One week after the third dose, the rabbits were bled. Two weeks after the bleeding they received a new dose and were bled one week later and so forth. All analyzes described in the following were performed with bleeding No . 7.
  • the immunizations were analyzed in immunoblottings, ELISA with fusion protein as the antigen and in competition ELISA with fusion protein coated microtiter plates.
  • Parasitized red blood cells were coated as a monolayer onto a slide with 12 wells. This was done by precoating the slide with the carbonate buffer pH 9.6 as mentioned for ELISA coating. Precoating was performed for 30 minutes. A drop of a suspension of parasites was placed over each well of the slide for 30 minutes . The slide was washed. Parasites were fixed by covering the wells with acetone and then airdried. Dilutions of the rabbit-anti-fusion protein antibodies were placed on the wells for 30 min. The wells were washed 4 times and fluorescence conjugated, porcine-anti-rabbit antibodies
  • DAKOPATTS F205 diluted 1:40 were applied to each well and incubated for 30 minutes after which period of time the slide was washed 4 times. The slide was then stained with 10 ⁇ g/ml ethidiumbromide and a cover was mounted. The slide was examined in an Olympus BH2
  • glutaraldehyde This fixation made the red blood cell membrane impermeable to antibodies in contrast to the fixation with acetone. No fluorescence was seen on glutaraldehyde fixed monolayers.
  • ELISAs Two types of ELISAs were used: a) coating of each well with 0.125 ⁇ g of fusion protein and direct reaction of rabbit serum with the fusion protein and subsequent demonstration of the binding of rabbit antibodies with porcine anti-rabbit immunoglobulin antibodies conjugated to peroxidase
  • mice were boosted 5 days before fusions. Culture supernatant from hybridomas were screened in the ELISA for detection of antibodies against the fusion protein. Three fusions have been performed resulting in detection of only a few clones with very low titers against the fusion protein.
  • Dilutions of hybridoma culture supernatant will be 1:4, detecting antibody conjugated to horseradish peroxidase (DAKOPATTS P260)
  • Antibodies from mice have been tested in immunoblotting showing reactivity with bands of molecular weights similar to the bands recognized by rabbit anti-fusion protein antibodies. Furthermore, antibodies from mice have been used as detecting antibodies in the antigen detecting ELISA, being able to recognize the varying amounts of culture supernatant being added as the specimen.
  • thiomersalatsodium The purpose of immunizing two of the monkeys with a vaccine supplemented with Freunds imcomplete adjuvant was to expose these two monkeys to the optimal stimulation of the immune systems, thereby serving as positive controls.
  • the vaccine was given subcutaneously in the posterior axillary line corresponding to the middle of the thorax.
  • the vaccine was given on day 0 of the immunization experiment, day 14 and day 28. Bleedings were performed 132 days before the start of the immunization
  • Serum from monkeys Nos. 867 and 865 diluted 1:200 has been tested in immunoblotting. They possess antibodies against the same parasite protein bands as the rabbits.

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Abstract

La présente invention se rapporte à un polypeptide comprenant une séquence d'acides aminés caractéristique tirée de l'antigène de Plasmodium falciparum GLURP, à un polypeptide qui est reconnu par un anticorps dressé contre ou réagissant avec un polypeptide comprenant ladite séquence d'acides aminés caractéristique et/ou un anticorps réagissant avec l'antigène GLURP natif, à une molécule d'acide nucléique (fragment d'ADN) codant pour ledit polypeptide, à un vecteur d'expression portant la molécule d'acide nucléique, à un organisme exprimant ladite molécule d'acide nucléique afin de produire ledit polypeptide, à un anticorps monoclonal dirigé contre ledit polypeptide, à un agent diagnostique comprenant ledit anticorps ou ledit polypeptide et destiné à être utilisé pour déterminer une affection au Plasmodium falciparum et pour diagnostiquer ainsi la malaria, ainsi qu'à l'utilisation dudit anticorps ou dudit polypeptide à des fins thérapeutiques, par exemple comme composant dans un vaccin. Ledit polypeptide comporte 3 unités de répétition uniques AENEESSLEE..., SEKSVSEPAEHVEIV et EEILPE.DKNEK...
EP89911024A 1988-09-16 1989-09-18 Antigene de malaria Withdrawn EP0434751A1 (fr)

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