EP0466912A1

EP0466912A1 - Glutathione-s-transferase sm26 of s. mansoni

Info

Publication number: EP0466912A1
Application number: EP91904781A
Authority: EP
Inventors: François TROTTEIN; André Capron; Doris Schmitt; Jean-Pierre Lecocq
Original assignee: Transgene SA
Current assignee: Transgene SA
Priority date: 1990-02-07
Filing date: 1991-02-06
Publication date: 1992-01-22
Also published as: KR920701445A; JPH04505402A; FR2657883A1; AU645786B2; CA2051695A1; WO1991012327A1; AU7333391A

Abstract

L'invention concerne une glutathion-S-transférase de Schistosoma mansoni, ayant un poids moléculaire de 26 Kd, appelée p26 ou Sm26 ainsi que les moyens de production de cette protéine sous une forme substantiellement purifiée.The invention relates to a glutathione-S-transferase 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.

Description

GLUTATHION-S-TRANSFERASE SM26 FROM S.MANSONI

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.

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.

It is already known that 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). However, this article does not explain how to obtain from the eluate, preparations containing each of the two glutathione-S-transferases in separate form. In addition, it is also known that Sm28, as obtained by recombinant DNA techniques, could be a candidate of choice as a schistosomiasis vaccine agent.

Finally, parallel studies on S. japonicum have similarly demonstrated similar antigens, i.e. Sp26 and Sp28, in the latter organism (Smith et al, Mol. Biochem. Parasitol. (1988) 27-249)

The DNA fragment coding for Sm26 having now been found, it is therefore possible to produce this protein by recombinant DNA techniques. This makes it possible inter alia to obtain Sm26 in the absence of Sm28 and in an amount sufficient to be purified.

Therefore, the present invention provides

(i) a protein whose amino acid sequence has at least 90%, advantageously at least 95% 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; said protein being in substantially pure form; as well as

(ii) a 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 ; said protein or said fragment being in substantially pure form.

This includes 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. By way of example, 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. To illustrate this latter case, 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. About 10 μl of a test solution, preferably prepared in a buffer medium close to neutrality , are added to 820 μl of a 50 mM potassium phosphate buffer solution; pH 6.5 which also contains 4.7 mM of reduced glutathione and 360 μM of l-chloro-2,4-dinιtrobenzene. In parallel, 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 μM / min / mg.

In another aspect, the invention also relates to:

i) an 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. By 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.

ii) 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.

iii) a cell which is transformed with an expression cassette according to the invention.

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.

iv) 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.

The technologies allowing the cloning and the expression of a foreign gene in prokaryotic or eukaryotic host cells are known to those skilled in the art. They will be illustrated in the examples below, it being understood that other vectors and other host cells can be used. Finally, the invention likewise relates to:

i) a pharmaceutical 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.

ii) 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.

iii) 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.

iv) the use of a protein according to the invention as a therapeutic agent intended to prevent or treat schistosomiasis.

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. In particular, 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. It is further indicated that, for use in a composition according to the invention, 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. Finally, a composition according to the invention may contain a vaccination adjuvant such as alum. Alternatively, 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.

In a last aspect of the invention, 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.

The examples below are intended to demonstrate other characteristics and advantages of the present invention, taking the following figures for reference.

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

1A. Anti-Sm26 antiserum preparation

A strain of the parasite S.mansoni is maintained in Biomphalaria glabrata

(aquatic snail) then in golden hamsters. The adult form of the parasite is collected from the blood taken from the portal vein of hamsters infected for 40 days. After washing in minimum Eagle medium (MEM), the worms are taken up in a buffer of formula: 10 mM potassium phosphate at pH 7.0; 1.15% KC1; 0.5 mM PMSF (Phenyl Methyl Sulphonyl Fluoride) and 1 mM EDTA (Ethylene Diamine TetraAcetic acid disodium knows). This preparation, subjected to ultrasound, is homogenized and then centrifuged for 20 min at 10,000 rpm at 4 ° C using a Sorvall HB-4 rotor. 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) .

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 μg of Sm26 in a total volume of 1 ml purified in the presence of complete Freund's adjuvant. Two weeks later, 20 μg of Sm26 are injected in the presence of incomplete Freund's adjuvant for subclavicular administration.

1B. Preparation of the complementary DNA library

Total 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 Gϋbler and Hoffman, Gene (1983) 25: 263. After protection of the internal EcoRI sites of the cDNAs, 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 β-galactosidase and are therefore placed under the control of the lactose promoter. Recombinant phages are packaged in vitro.

1 C. Selection of a cDNA coding for Sm26

1.5. 10 ₆ phages independent of the complementary DNA library are tested by the method described by Huynh et al., DNA cloning, a Practical Approach, (1985) Glover DM, ed., _ 1:49, modified as follows: a sample from this lambda gtll library is inoculated on the E. coli Y1090 strain at a dilution representing 5000 phages per 85 mm diameter dish and incubated at 42 ° C. The plaques are transferred for 5 h at 37 ° C. onto nitrocellulose filters (Schleicher and Schuell) previously impregnated with isopropyl-β-D-thiogalactopyranoside (IPTG) at 10 mM; IPTG induces the expression of the fusion protein which is under the control of the lac promoter. 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.

Four phage clones capable of expressing a protein reacting with the anti-Sm26 antiserum are harvested separately. Their DNAs are purified and digested with EcoRI. Then the fragments from the digestion are separated by electrophoresis on agarose gel. One of the cDNA inserts, of sufficient size to code for a complete Sm26, is electroeluted by the method of Geneclean (Bio 101 Inc. La Jolla, CA) then subcloned in the EcoRI site of the multiple insertion region of the vector M13TG130 (Kieny et al, Gene (1983) 26: 91). The nucleotide sequence of this cDNA is established by the method of Sanger et al, PNAS (1977) 74: 5463. The study of the sequence makes it possible to establish that this particular cDNA codes well for a 26 Kd protein. Since this cDNA does not have the base A of the ATG initiation codon, it must therefore be added. In addition, it is desired to replace the EcoRI site with a BglII site. These two operations are carried out jointly by site-directed mutagenesis using the oligonucleotide OTG2467 of sequence:

5 '-GCTCTCCCGGAGATCTATGGC ACCTAAGTT-3'.

The vector M13TG4116 is thus obtained. The cDNA sequence is presented in the sequence identifier.

EXAMPLE 2 Production and purification of Sm26

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. In parallel, 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. In this latter construction, 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.

At the end of culture, 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 μg / ml of lysosyme and 0.1% of newt

X100 (Sigma).

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. After intensive washing with this same buffer, 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. All of the eluate is recovered, neutralized using 0.5 M potassium phosphate pH 7, then dialyzed overnight at 4 ° C. against a dialysis buffer of formula: 10 mM potassium phosphate pH 7; 1 mM EDTA. The dialysate is finally concentrated in a volume of approximately 2 ml with aquacid -Q (Calbiochem - Behring Corp. CA).

The purity of the preparation thus obtained is checked after electrophoresis of a polyacrylamide gel sample. As shown in Figure 2, the appearance of a single band of approximately 26 Kd indicates that Sm26 is obtained in substantially pure form.

On the other hand, the glutathione-S-transferase activity of the preparation thus purified is controlled as follows: 10 μl of the preparation are added to 820 μl of a 50 mM potassium phosphate buffer solution; pH 6.5; which also contains 4.7 mM of reduced glutathione and 360 μM 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 μM / 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.

EXAMPLE 3 Demonstration of the Sm26 Immunoprotective Effect

3 A. Experiment 1

8 week old male Fisher rats (Iffa-Credo) are divided into 5 groups of 4 to

7 rats each. The rats receive 25 μg of Sm26 subcutaneously as obtained in Example 2 (group 1), 25 μg of Sm28 purified from adult worms (group 2) or 25 μg 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). In parallel, the rats of group 4 receive only 1.25 mg of aluminum hydroxide adjuvant in the same volume. Finally, the rats of group 5 receive nothing. For each of groups 1 to 4, the corresponding treatment is repeated 15 days after the first injection.

30 days after the second 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

(adjuvant control witness). The result of group no. 1, although lower than that of group no. 2, is quite significant due to the low standard deviation (p = 0.03).

3B. Experiment 2

A new experiment, the experimental conditions of which are in all respects similar to those described under 3A, is carried out independently. The group of rats immunized with Sm26 show a reduction in parasite load by 25%.

SEQUENCE IDENTIFIER

Subject: 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.

GCA CCT AAG TTC GGT TAT TGG AAA GTC AAA GGC CTT GTA CAA CCA 45 Ala Pro Lys Phe Gly Tyr Trp Lys Val Lys Gly Leu Val Gin Pro 1 5 10 15

ACT CGA CTT CTT TTG GAA CAC CTT GAA GAA ACT TAT GAG GAA CGT 90 Thr Arg Leu Leu Leu Glu His Leu Glu Glu Thr Tyr Glu Glu Arg

20 25 30

GCG TAT GAT CGC AAT GAA ATC GAT GCC TGG AGC AAC GAT AAA TTT 135 Ala Tyr Asp Arg Asn Glu Ile Asp Ala Trp Ser Asn Asp Lys Phe

35 40 45

AAA TTA GGC CTG GAG TTC CCA AAT CTT CCT TAT TAT ATT GAT GGT 180 Lys Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly

50 55 60

GAT TTT AAA TTA ACA CAA TCT ATG GCT ATC ATA CGT TAT ATA GCT 225 Asp Phe Lys Leu Thr Gin Ser Met Ala Ile Arg Arg Ile Ala

65 70 75

GAC AAA CAC AAC ATG TTG GGG GCT TGT CCA AAA GAA CGT GCG GAA 270 Asp Lys His Asn Met Leu Gly Ala Cys Pro Lys Glu Arg Ala Glu

80 85 90

ATT TCG ATG CTT GAA GGA GCG GTT TTG GAT ATT AGG ATG GGT GTT 315 Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg Met Gly Val

95 100 105

TTA AGA ATC GCA TAC AAT AAG GAA TAT GAA ACC CTC AAA GTT GAT 360 Leu Arg Ile Ala Tyr Asn Lys Glu Tyr Glu Thr Leu Lys Val Asp

110 115 120 TTT CTC AAC AAA CTT CCT GGG AGG CTG AAA ATG TTC GAA GAT CGT 405 Phe Leu Asn Lys Leu Pro Gly Arg Leu Lys Met Phe Glu Asp Arg 125 130 135

TTG TCT AAC AAA ACT TAT TTG AAC GGT AAT TGT GTA ACT CA .CCT 450 Leu Ser Asn Lys Thr Tyr Leu Asn Gly Asn Cys Val Thr His Pro 140 145 150

GAC TTT ATG TTA TAC GAT GCC CTT GAT GTG GTT TTA TAC ATG GAG 495 Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp 155 160 165

TCA CAG TGC TTG AAC GAG TTT CCA AAA TTA GTT TCT TTC AAA AAG 540 Ser.Gin Cys Leu Asn Glu Phe Pro Lys Leu Val Ser Phe Lys Lys 170 175 180 f TGT ATT GAA GAT TTA CCA CAA ATC AAG AAC TAC TTA AAT TCT AGC 585

Cys Ile Glu Asp Leu Pro Gin Ile Lys Asn Tyr Leu Asn Ser Ser

185 190 195

AGG TAC ATA AAA TGG CCT CTG CAA GGT TGG GAT GCC ACG TTT GGT 630 Arg Tyr Ile Lys Trp Pro Leu Gin Gly Trp Asp Ala Thr Phe Gly 200 205 210

GGT GGA GAT ACT CCT CCA AAA 651

Gly Gly Asp Thr Pro Pro Lys 215

Claims

- 13 - CLAIMS

1. A 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; said protein being in substantially pure form.

2. A 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 ; said protein being in substantially pure form.

3. A DNA fragment coding for a protein according to claim 1 or for a protein or a fragment thereof according to claim 2.

4. An expression cassette which comprises a DNA fragment according to claim 3 and the elements necessary for the expression of said DNA fragment in a host cell.

5. A cell transformed with an expression cassette according to claim 4.

6. A cell according to claim 5, which is of the species E. coli.

7. A method of preparing a protein according to claim 1 or 2 which comprises the act of cultivating a cell according to claim 5 or 6 and harvesting said protein from the culture.

8. Application of a protein according to claim 1 or 2 as an agent having glutathione transferase activity.

9. A pharmaceutical 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 l sequence identifier, starting with the alanine residue in position 1 and ending with the lysine residue in position 217. - 14 -

10. A pharmaceutical composition intended for the prevention or treatment of schistosomiasis which comprises, as a 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.