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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
  • C07K14/43559—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from trematodes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • 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

• the present invention relates to a glutathione S-transferase (GST) from Schistosoma mansoni, having a molecular weight of 26 Kd, called p26 or Sm26, as well as the means for producing this protein in a substantially purified form.
• GST glutathione S-transferase
• Schistosomes are worms that are the agents of a parasitic disease in the tropics and subtropics, called schistosomiasis (or bilharziasis). This disease affects around 200 to 400 million people. There is a drug effective against the adult parasite, praziquantel, but its cost is too high to consider its widespread use in developing countries. The only serious hope for preventing the disease to date lies in the development of an effective vaccine against the parasite.
• S. mansoni has at least two glutathione-S-transferases; one with a molecular weight of 26 Kd, the other, predominant, with a molecular weight of 28 Kd, called p28 or Sm28.
• the existence of these two proteins has been demonstrated on SDS-polyacrylamide gel after electrophoresis of the sample of an eluate obtained by affinity chromatography of an extract of S. mansoni on a glutathione-agarose column (Tiu et al, Parasite Immunol., (1988) OJ: 693).
• this article does not explain how to obtain from the eluate, preparations containing each of the two glutathione-S-transferases in separate form.
• Sm28 as obtained by recombinant DNA techniques, could be a candidate of choice as a schistosomiasis vaccine agent.
• Sm26 as well as analogues thereof. Like Sm26, these analogs could be characterized by glutathione-S-transferase activity or by the ability to induce in an mammal an immunological response which protects this mammal against infection by schistosomes.
• the analog (45-80) is cited, starting with the lysine residue in position 45 and ending with the histidine residue in position 80.
• a protein according to the invention in particular the Sm26 protein whose amino acid sequence is as shown in the sequence identifier can exist in monomeric or multimeric form.
• the dimeric form which may advantageously be maintained by inter-chain disulphide bridges is cited by way of example.
• Glutathione-S-transferase activity can be demonstrated by the method described by Hahig & Ja oby, Methods in Enzymology, 77: 398.
• a test solution preferably prepared in a buffer medium close to neutrality
• 820 ⁇ l of a 50 mM potassium phosphate buffer solution pH 6.5 which also contains 4.7 mM of reduced glutathione and 360 ⁇ M of l-chloro-2,4-din ⁇ trobenzene.
• a negative control is carried out.
• the appearance of the reaction product is followed by spectrophotometry at 340 nm until complete transformation of the substrate. Glutathione-S-transferase activity is given in ⁇ M / min / mg.
• the invention also relates to:
• isolated DNA fragment which codes for a protein according to the invention.
• a particular example of a DNA fragment according to the invention is shown in the sequence identifier.
• isolated DNA fragment is meant a DNA fragment which is no longer associated with the regions which control its expression in the organism from which it originates, that is to say in the present case S.mansoni.
• an expression cassette which comprises a DNA fragment according to the invention as well as elements necessary for its expression (transcription and translation) in a host cell.
• These elements are essentially an appropriate transcription promoter as well as the start and end of translation codons. In some cases, it is also advantageous to add a transcription terminator.
• This expression cassette can either be integrated into the genome of the cell, or carried by an expression vector suitable for autonomous replication, e.g. a plasmid.
• a process for preparing a protein according to the invention which comprises the act of cultivating a cell according to the invention and of harvesting said protein from the culture.
• composition intended for the prevention or treatment of schistosomiasis which comprises, as therapeutic agent, at least one protein whose amino acid sequence has at least 90% homology with the sequence as shown in the sequence identifier, starting with the alanine residue in position 1 and ending with the lysine residue in position 217.
• a pharmaceutical composition intended to prevent or treat schistosomiasis • which comprises, as therapeutic agent, at least one protein whose amino acid sequence is as shown in the sequence identifier, starting with the alanine residue in position 1 and ending with the lysine residue in position 217, or a fragment of said protein containing at least 22 amino acids.
• a method for preventing or treating schistosomiasis which comprises the act of administering a therapeutically effective amount of a protein according to the invention to a patient in need of such treatment.
• a protein according to the invention or a fragment thereof has a therapeutic activity, for example a vaccine activity, and therefore is useful as a pharmaceutical product. This activity can be demonstrated in a test identical or similar to that described in Example 3.
• a pharmaceutical composition according to the invention can be manufactured in a conventional manner.
• a protein according to the invention or a fragment of said protein is combined with a diluent or a support which is acceptable from a pharmaceutical point of view.
• the protein may advantageously be in monomeric or dimeric form, the latter form being particularly advantageous.
• a composition according to the invention may also contain other compounds such as other glutathione-S-transferases from schistosomes eg Sp26, Sp28, Sm28, preferably in dimeric form, or one of the analogs thereof.
• a composition according to the invention may contain a vaccination adjuvant such as alum.
• this adjuvant can be added to a composition according to the invention just before use.
• a composition according to the invention can be administered by any conventional route in use in the field of vaccines, in particular by the subcutaneous route, for example in the form of a solution or suspension for injection. The administration can take place in single dose or repeated one or more times after a certain interval of interval. The appropriate dosage will vary depending on various parameters, for example, the individual being treated and the mode of administration.
• a protein according to the invention or a fragment thereof can in particular be useful as an agent exhibiting glutathione transferase activity. Therefore, it can be used for example in bioconversion processes.
• Figure 1 shows the vector pTG959.
• Figure 2 shows a polyacrylamide-SDS gel revealed by staining with Coomassie blue after electrophoresis.
• Columns a, b and c correspond respectively to the non-purified soluble and insoluble fractions of the TGE901 / pTG4l70 extracts after induction for 5 h at 42 ° C. and to the preparation purified on a glutathione-agarose column.
• Figure 3 compares the amino acid sequences of the Sm26 and Sj26 proteins. In these, spaces are schematically introduced so as to obtain an optimal alignment.
• EXAMPLE 1 Cloning of a complementary DNA coding for Sm26
• the supernatant is recovered and then applied to a gluthation-agarose column (Sigma Chemical Co, St. Louis, Mo) equilibrated with the buffer described above. After intensive washing with this same buffer, the fixed fraction is eluted with an elution buffer (50 mM Tris-HCl, pH 9.1, 7 mM reduced gluthation and 0.1 mM dithiotreitol). The eluted fraction is then neutralized using the 0.5 M potassium phosphate buffer, pH 7, then dialyzed against 10 mM potassium phosphate, pH 7 and finally concentrated with aquacid II (Calbiochem -Behring Corp, CA) .
• an elution buffer 50 mM Tris-HCl, pH 9.1, 7 mM reduced gluthation and 0.1 mM dithiotreitol.
• the eluted fraction is then neutralized using the 0.5 M potassium phosphate buffer, pH 7, then dialyzed against 10 mM potassium phosphate, pH 7 and finally concentrated with aqua
• the total S-mansoni gluthation S-transferases thus purified are fractionated on a preparative polyacrylamide gel (13%) according to the technique described by Laemmli et al, Nature, (1970) 227: 680. After staining with Coomassie blue, the strip migrating at 26 kDa is recovered. Sm26 is electro-eluted according to the protocol described by Balloul et al., Mol. Biochem. ParasitoL, (1985) Yh 105. It is dialyzed against water and then concentrated using aquacid II.
• Rabbits receive by multi-site intradermal injection (40 points) 100 ⁇ g of Sm26 in a total volume of 1 ml purified in the presence of complete Freund's adjuvant. Two weeks later, 20 ⁇ g of Sm26 are injected in the presence of incomplete Freund's adjuvant for subclavicular administration.
• RNA from adult schistosomes is extracted by the method described by Chirgwin et al., Biochemistry (1979) Jjî: 5294 and modified by Gausz et al., Mol. Biochem. ParasitoL, (1983) 7: 293.
• the poly A + RNA is obtained by affinity chromatography on an oligo (dT) cellulose column 12-18 (Collaborative Research, Lexington, MA).
• a complementary DNA library expressed in the lambda gt 11 vector is constructed using the Amersham kit as follows. From purified poly A + RNAs, complementary DNAs (cDNAs) are synthesized according to the method of G ⁇ bler and Hoffman, Gene (1983) 25: 263.
• EcoRI adapters are attached to the cDNA fragments. These are then digested with EcoRI and the excess adapters are removed by column chromatography. These cDNA fragments are inserted into the EcoRI site of phage lambda gt 11 (Young and Davis, 1983). The EcoRI inserts are thus fused with the sequence coding for ⁇ -galactosidase and are therefore placed under the control of the lactose promoter. Recombinant phages are packaged in vitro.
• IPTG isopropyl- ⁇ -D-thiogalactopyranoside
• the filters are incubated in the presence of anti-p26 rabbit antiserum (previously adsorbed on an extract of E.colQ antigens.
• the fixed antibodies are recognized by a second anti-rabbit IgG antibody labeled with biotin; a streptavidin -peroxidase then binds to biotin, and this complex is revealed by a reaction of peroxidase on a colored reagent called "HRP color development reagent", marketed by Bio Rad.
• This antibody detection makes it possible to identify the recombinant phages whose cDNA insert directs the synthesis of a protein or a protein fraction carrying epitopes recognized by specific anti-Sm26 antibodies.
• the vector M13TG4116 is thus obtained.
• the cDNA sequence is presented in the sequence identifier.
• the vector M13TG4116 is digested with EcoRI and BglII, and the DNA fragments are separated by electrophoresis on agarose gel.
• the 0.75 Kb fragment comprising the Sm26 cDNA is eluted by the Geneclean method.
• the vector pTG959 described in EPA 292 404 and shown schematically in Figure 1 is digested with EcoRI and BglII. These cuts result in the loss of a fragment containing the 5 'part of the lacZ gene.
• the 0.75 Kb fragment is ligated into the vector pTG959 as previously prepared to give the vector pTG4170.
• the DNA fragment coding for Sm26 is therefore placed downstream and under the control of the pL promoter of phage lambda which is inducible at 42 ° C.
• E.coli TGE901 (F-sup-his ilv bio (lambda-ci 857 delta-Bam delta-Hl)) transformed by the plasmid pTG4170 is cultured in 11 LB medium - ampicillin. The culture is continued, firstly at 30 ° C until an OD ⁇ of 0.2 and secondly at 42 ° C for 5 hours.
• the cells are harvested by centrifugation and then lysed both by sonication and by enzymatic treatment in an equilibration buffer of formula: 10 mM potassium phosphate, pH7; 1.15% w / v KCI; 0.5 mM Phenyl Methyl Sulphonyl Fluoride; 1 mM EDTA, additionally containing 100 ⁇ g / ml of lysosyme and 0.1% of newt
• the lysate is then centrifuged at 10,000 rpm at 4 ° C for 20 min.
• the supernatant is recovered and then deposited in a continuous stream (30 ml / h) on a glutathione-agarose column (Sigma Chemicals Co., St Louis, Mo) previously equilibrated with the equilibration buffer described above.
• the fixed fraction is eluted with 10 ml of elution buffer of formula: 50 mM Tris-HCl, pH9.1; 7 mM glutathione in reduced form; 0.1 mM dithiothreitol.
• the glutathione-S-transferase activity of the preparation thus purified is controlled as follows: 10 ⁇ l of the preparation are added to 820 ⁇ l of a 50 mM potassium phosphate buffer solution; pH 6.5; which also contains 4.7 mM of reduced glutathione and 360 ⁇ M of l-chloro-2,4-dinitrobenzene. In parallel, a negative control is carried out. The appearance of the reaction product (yellow coloring) is followed at 340 nm until complete transformation of the substrate. The activity measured is of the order of 30 to 50 ⁇ M / min / mg.
• the method of preparation of Sm26 previously described makes it possible to obtain approximately 1 to 2 mg of purified protein from a liter of culture.
• the rats receive 25 ⁇ g of Sm26 subcutaneously as obtained in Example 2 (group 1), 25 ⁇ g of Sm28 purified from adult worms (group 2) or 25 ⁇ g of bovine serum albumin (group 3).
• Each antigen is injected in a volume of 1 ml in the presence of 1.25 mg of aluminum hydroxide adjuvant (Serva alum gel).
• the rats of group 4 receive only 1.25 mg of aluminum hydroxide adjuvant in the same volume.
• the rats of group 5 receive nothing.
• the corresponding treatment is repeated 15 days after the first injection.
• the rats of each group are anesthetized, then infested by transcutaneous passage of 1000 cercariae.
• 21 days after the infestation the livers are ligated and perfused with physiological fluid containing 1% heparin and 0.1% Tween. This makes it possible to recover the worms which are then counted under the binocular magnifier.
• the results are presented in the table below.
• Group n ° 2 (positive control) and group n ° 1 show a reduction in parasitic load of 45% and 16% respectively compared to group n ° 4
• Sm26 The amino acid sequence of the glutathione-S-transferase of 26 Kd from S.mansoni, called Sm26 as well as the nucleotide sequence of the complementary DNA fragment coding for the protein cited above.

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• 1990
  • 1990-02-07 FR FR9001403A patent/FR2657883A1/fr active Pending
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