WO1991009621A1 - PEPTIDE FRAGMENT COMPRISING A SEQUENCE FROM THE 28kDa PROTEIN OF SCHISTOSOMA MANSONI, AND VACCINATING AND/OR THERAPEUTIC COMPOSITIONS COMPRISING SAID FRAGMENT - Google Patents

Abstract

Peptide fragment obtained from the 28 kDa protein of S. mansoni, and vaccinating and/or therapeutic compositions comprising said peptide fragment, optionally in association with other suitable substances. Said peptide fragment includes an aminoacid sequence from the 28 kDa protein of Schistosoma mansoni, represented 2n times in said peptide fragment, n being a number higher than 1. Said sequence is further characterized in that it comprises at least one epitope which induces a specific T lymphocyte response, and at least one epitope which induces a specific B lymphocyte response.

Description

PEPTIDE FRAGMENT COMPRISING A SEQUENCE FROM THE 28 kDa PROTEIN OF SCHISTOSOMA MANSONI AND VACCINATING AND / OR THERAPEUTIC COMPOSITIONS COMPRISING SAID FRAGMENT. The present invention relates to a peptide fragment obtained from the protein 28 kDa of S. mansoni, as well as to vaccinating and / or therapeutic compositions comprising said peptide fragment, optionally combined with other suitable substances. BALLOUL et al. [MOLECULAR AND BIOCHEMICAL

PARASITOLOGY, 12, (1985), p. 105-114] have described the in vitro synthesis of an antigen constituted by a polypeptide having a molecular weight of 28 kDa and an isoelectric point between 6.3 and 6.8, which constitutes an in vitro translation product total Schistosoma mansoni RNA. These Authors have continued their research with the aim of isolating from this 28 kD polypeptide, and identifying the epitope or peptide epitopes responsible for the antigenic properties. It is known to analyze the peptides which constitute proteins isolated from SDS gels, by partial digestion of said proteins with a protease in a buffer containing SDS; stable partial digestion products are obtained, which are composed of numerous peptides whose molecular weights are sufficiently high so that it is possible to separate them into SDS acryla ide gels at 15% [CLEVELAND et al. , THE JOURNAL OF BIOLOGICAL CHEMISTRY, 252, p. 1102-1106, (1977)]. As the inventors have shown précédem¬ ment, particularly in the French Patent ^No. 2,601,037, they have sought to isolate, from 28 kD protein of S. mansoni, the peptide with activity antigé- anti-Schistoso e that presents the protein 28 kD, and have applied for this purpose the techniques of isolation of peptide fragments by proteolysis described in the prior art. Continuing their work in this direction, they more precisely highlighted the antigenic determinants involved in protective immunity.

The article published in J. Immunol., 1988, 141, 5, 1687-1694 describes in particular three peptides derived from the primary sequence of the P28 antigen of Schistosama manson. These three peptides respectively comprise the sequences 24-43, 115-131 and 140-153 of said P28 antigen. This article also specifies that the peptides comprising the sequences 24-43 and 115-131 contain major epitopes for IgG and not for IgE.

The specific IgGs obtained after injection of the P28 antigen or of peptide 24-43 coupled with the tetanus toxoid to Fischer rats are essentially IgG2a and to a lesser extent IgG_; these specific antibodies are cytotoxic in vitro against schistosomules in the presence of eosinophils as effector cells.

The article published in J. Immunol., 1989, 142. 4, 1342-1350 highlights, for its part, the immune protection of mice vaccinated with the P28-1 antigen of Schistosoma mansoni. This P28-1 antigen corresponds to the 172 amino acids of the COOH end of the native 28 kDa protein. It is in particular specified that the T lymphocytes of mice immunized with the recombinant P28-1 antigen are stimulated in vitro by schistosomal antigens of different stages of development and by three synthetic peptides derived from the P28-1 antigen. The most effective stimulation is obtained with peptide 24-43. The use of two fragments of this peptide has shown that the specificity with respect to T lymphocytes is provided by the NH2 terminal sequence of said peptide.

However, these peptides have the disadvantage of not being sufficiently immunogenic. In fact, an ideal vaccine against bilharziasis must be strongly immuno- gene and be able to induce both specific immunity against the schistosome for both T cells and B cells.

The inventors, continuing their work in this field, have developed peptide fragments which better meet the needs of the practice than the peptide fragments of the prior art, in particular in that they are both immunogenic and protective and thus make it possible to obtain a far more protective vaccinating composition.

The subject of the present invention is a peptide fragment, characterized in that it comprises an amino acid sequence derived from the 28 kDa protein of Schistosoma mansoni, represented 2 ⁿ times in said peptide fragment, n being a higher number at 1, which sequence is further characterized in that it comprises at least one epitope which induces a specific response of T lymphocytes and at least one epitope which induces a specific response of B lymphocytes, represented 2 ⁿ times. The 2 ⁿ times representation of the sequence according to the invention unexpectedly results in a markedly increased induction of both an immune response T (cellular response) and an immune response B (humoral response). According to an advantageous embodiment of said peptide fragment, n is advantageously a number comprised between 2 and 10.

According to a preferred arrangement of this embodiment, n is equal to 3. According to another advantageous embodiment of said peptide fragment, said sequence originating from the 28 kDa protein is the sequence 115-131 of said native 28 kDa protein or recombined.

The sequence 115-131 includes the following amino acids: Lys-Pro-Gln-Glu-Glu-Lys-Glu-Lys-Ile-Thr-Lys- Glu-Ile-Leu-Asn-Gly-Lys-COOH According to an advantageous arrangement of this embodiment, the sequence 115-131 of the protein 28 kDa is represented 8 times (n = 3).

Such a so-called multiantigenic peptide fragment has been called the "octopus" fragment by the inventors.

This peptide fragment called "octopus" notably has the advantage of inducing both a humoral response and a cellular response, while the original peptide induces practically no humoral response.

The process for the preparation of said pep¬ tic fragment comprises the recurrent synthesis of the sequence derived from the 28 kDa protein as defined above on a matrix, carrying 2 ⁿ functional groups, as described in JP TAM (Proc. Natl. Acad. Sci. USA, 1988, £ 5., 5409-5413).

The present invention further relates to an immunogenic and protective composition, characterized in that it comprises at least one peptide fragment as defined above, optionally combined with at least one other suitable substance and / or with an adjuvant appropriate.

Such a preparation unexpectedly exhibits significant immunogenic and protective properties.

In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention.

It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation. Example 1: Process for the preparation of the peptide fragment in accordance with the invention.

The synthesis was carried out manually on a t-butyloxycarbonyl (Boc) β-Alanine benzhydrilamide resin loaded at 0.3 mmol / g. The assembly of the octovalent matrix was carried out according to the protocol described in 1988 by the team of Tam (Proc. Natl. Acad. Sci. USA, ϋϋ, 5409-5413): after deprotection of the t-butyloxycarbonyl group by the acid trifluoroacetic at 50% in methylene chloride and neutralization by diisopropylethylamine, the synthesis of the first level, to obtain Boc-Lys (Boc) -β Ala-BHA resin, is carried out by coupling of 2.5 molar excess of Boc-Lys (Boc) introduced in the form of the dicyclohexylamine salt according to the BOP procedure [CASTRO et al., SYNTHESIS, 1976 751-752: Peptide coupling reagents; VI A novel cheaper preparation of benzotriazolyloxytriε (dimethylamino) -hexafluorophosphate (BOP reagent)] in dimethylformamide (DMF), followed by recoupling in methylene chloride. The second and third levels were synthesized according to the same protocol, considering a charge multiplied by 2 then by 4 respectively, to obtain an octavalent Boc-Lys (Boc) -matrix containing 8 functional amino groups. Thereafter, the synthesis of the peptide antigen is carried out according to the usual Boc / TFA scheme, the protections of the side chains are identical to the protections used in the synthesis of the monomer. The couplings are carried out according to the BOP procedure in the DMF, followed by one or two recoupling, if necessary, in an NMP-DMSO solvent mixture [Curdy, Otteson, Applied Biosystems User Meeting, St Louis, May 1987: the use of N-methylpyrrolidone-dimethyl tallow oxide as a coupling mixture in solid phase synthesis]. The monitoring of the synthesis is carried out by means of the Kaiser test with ninhydrin. The sequence of the 115-131 antigen is as follows: Lys-Pro-Gln-Glu-Glu-Lys-Glu-Lys-Ile- Thr-Lys-Glu-Ile-Leu-Asn-Gly-Lys. At the end of the synthesis, the Boc group is cleaved at 50% TFA, then the "octopus 115-131" is released from its support and simultaneously deprotected by treatment with anhydrous hydrofluoric acid (HF) or trifluoromethane sulfonic acid in dimethyl sulfide, to avoid harmful reactions catalyzed by strong acids. An octameric structure is obtained, as visible in FIG. 1. The crude product is purified by extensive dialysis on a YM5 membrane (Amicon) against an HCl solution pH3. Control by amino acid analysis after total acid hydrolysis gives a satisfactory amino acid composition. Example 2: Vaccinating role of the composition in accordance with the invention. The synthetic peptides, derived from the primary sequence of the recombinant P28 protein, were synthesized and the presence of T and B cells was examined in rats, mice, baboons and humans.

The peptide comprising amino acids 115-131 contains the major epitopes of T and B cells.

A multiantigenic peptide 115-131 ("octo¬ pus") was prepared as specified in Example 1 and used as an immunogenic composition in rats, mice and baboons. In the three experimental models, the "octopus 115-131" induces both a humoral response and a cellular response towards the recombinant P28 protein or the native P28 protein.

In addition, prior immunization with "octopus 115-131" before an injection of P28 protein leads to an increase in the order of 4 to 10 times in the proliferation of T cells and in the production of antibodies.

Active immunization of rats with this peptide fragment according to the invention, before infection, allows significant protection of the order of 40 to 50%. The protection obtained is shown in FIG. 2, which represents a histogram in which there are the number of worms present with in column (1), a negative control (injection of ovalbumin) and in column (2), the protection obtained after immunization with octopus 115-131.

The rat immunization protocol is as follows:

. on day 0 (D0), Fischer rats are immunized using 100 μg "of octopus 115-131" by adjuvanting complete with Freund, by subcutaneous route, at the base of the tail of aged rats seven weeks;

. at D7 (7th day), a first booster is performed, with 50 μg of "octopus 115-131" by Incomplete Freund's adjuvant (AIF), by subcutaneous route;

. on D14 (14th day), a second booster is performed, with 50 μg of "octopus 115-131" by Incomplete Freund's adjuvant (AIF), by subcutaneous route;

. on D24 (24th day), a third booster is carried out, with 50 μg of "octopus 115-131" by Incomplete Freund's adjuvant (AIF), by subcutaneous route; . at D30 (30th day), that is to say: the draining nodes are recovered, when one wishes to carry out a lymphocyte specificity test;

- Rats are infested with 1500 furcocercariae, when one wants to carry out protective experiments. Example 3: Comparison between a sequence according to the invention (represented 2 ⁿ times of the same sequence) with the original sequence. at. Immunization:

The immunization of rats is carried out as specified in Example 2, either with the octopus 115-131, or with the peptide 115-131. The following Table I shows the serum titers obtained with the two above-mentioned sequences.

The titer corresponds to the dilution of serum necessary to obtain an optical density of 1. TABLE I

This Table I shows that the native peptide induces practically no humoral response. b. T cell response:

The response obtained is shown in FIG. 3, which represents a histogram in which is represented the proliferative response in vitro of T lymphocytes of Fisher rats immunized with the protein 28 kDa (also called Sm28 Gst, by the inventors), after incubation with protein 28 kDa (column 1), total adult worm anti¬ genes (S AP, column 2), ovalbumin (column 3), octopus 115-131 (column 4) and native peptide 115- 131 (column 5), all of these molecules being at a concentration of 80 μg / ml.

As is apparent from the above, the invention is in no way limited to those of its embodiments and of application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the scope or the scope of the present invention.

Claims

CLAIMS 1 ^* ) Peptide fragment, characterized in that it comprises an amino acid sequence derived from the protein 28 kDa of Schistosoma mansoni, represented 2 ⁿ times in said peptide fragment, n being a number greater than 1, which sequence is further characterized in that it comprises at least one epitope which induces a specific response of T lymphocytes and at least one epitope which induces a specific response of B lymphocytes 2 ^* ) Fragment according to claim 1, charac¬ terized in that n is advantageously a number between 2 and 10. 3 ") Fragment according to claim 2, characterized in that n is equal to 3. 4 ^* ) Fragment according to any one of claims 1 to 3, characterized in that said sequence derived from the 28 kDa protein is the sequence 115-131 of said 28 kDa protein. 5 ^* ) Fragment according to claim 4, charac¬ terized in that the sequence 115-131 of the protein 28 kDa is represented 8 times (n = 3). 6 ') Immunogenic and protective composition, ca¬ characterized in that it comprises at least one peptide fragment according to any one of claims 1 to 5 optionally associated with at least one other suitable substance and / or with a suitable adjuvant.